NSP4 inhibitors and methods of use

ABSTRACT

The invention provides NSP4 inhibitors (such as anti-NSP4 antibodies) and methods of using the same.

CROSS REFERENCES TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.15/094,843, filed Apr. 8, 2016, now U.S. Pat. No. 9,975,963, issued onMay 22, 2018, which is a continuation of International Application No.PCT/US2014/060182, filed internationally on Oct. 10, 2014, which claimspriority to and the priority benefit of U.S. Provisional ApplicationSer. No. 61/890,147, filed Oct. 11, 2013; 61/893,059, filed Oct. 18,2013; and 62/053,052, filed Sep. 19, 2014; each of which is incorporatedherein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 146392022740SEQLIST.txt,date recorded: Oct. 8, 2014, size: 63 KB).

FIELD OF THE INVENTION

The present invention relates to NSP4 inhibitors and methods of usingthe same.

BACKGROUND

Neutrophil serine proteases are a family of effector molecules of theinnate immune system that are important for protecting against invadingpathogens. Members of this trypsin-fold protease family, neutrophilelastase (NE), cathepsin G (CG), and proteinase 3 (PR3), not only playcritical roles in neutrophil-mediated clearance of invading microbes(Reeves et al., Nature, 2002, 416:291-297; Weinrauch et al., Nature,2002, 417:91-94; Belaaouaj et al., Nat Med, 1998, 4:615-618) andinflammation (Pham et al., Nat Rev Immunol, 2006, 6:541-550) but canalso participate in the pathogenesis of various diseases (Magrone etal., Curr Pharm Des., 2012, 18(12):1609-19 and Pham et al., Int BiochemCell Biol., 2008, 40(6-7):1317-1333). Recently, the serine proteasePRSS57, originally discovered by yeast signal trap screening andcomputational mining of human cDNA libraries (Clark et al., Genome Res.,2003, 13:2265-2270), was identified as the fourth NSP member andsubsequently referred to as neutrophil serine protease 4 (NSP4) (Pereraet al., Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera et al., JImmunol., 2013). Remarkably, NSP4 is highly conserved from bony fishesto human and predates the emergence of other NSPs, indicating that NSP4likely plays fundamental roles in neutrophil biology (Perera et al.,Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera et al., Expert RevClin Immunol, 2012, 8:501-503).

The relatively broad substrate specificities of NE, CG, and PR3 are wellunderstood based on the detailed knowledge of their active sitestructures (Navia et al., Proc Natl Acad Sci USA, 1989, 86:7-11; Hof etal., EMBO J, 1996, 15:5481-5491; Fujinaga et al., J Mol Biol, 1996,261:267-278.). However, NSP4 poses a conundrum in that, like trypsin, itcleaves substrates after arginine residues (Perera et al., Proc NatlAcad Sci USA, 2012, 109:6229-6234; Perera et al., J Immunol., 2013), butparadoxically has a primary sequence that predicts a very differentelastase-like active site with preference for small aliphatic aminoacids and is seemingly incompatible with the long P1-arginine sidechain. Due to NSP4's long evolutionary lineage, and its distinctiveactive site, it is possible that NSP4 is an important protease forneutrophil function and may contribute to neutrophil-mediated disease ordisorders. However, relative to other members of the neutrophil serineprotease family, the role of NSP4 in neutrophil-mediated diseases, suchas arthritis, is unknown. For instance, the combined deficiencies of NEand CG was required to confer full protection in the mouse collagenantibody-induced arthritis model (Adkison et al., J Clin Invest, 2002,109:363-371). NE and CG are also each capable of processing andactivating IL-33 (Lefrançais et al., Proc Natl Acad Sci USA, 2012,109:1673-1678), which is a pro-inflammatory cytokine that promotesinflammatory arthritis (Xu et al., Proc Natl Acad Sci USA, 2008,105:10913-10918). Similarly, the combined ablation of NE and PR3 wasrequired to prevent the inactivation of progranulin (Kessenbrock et al.,J Clin Invest, 2008, 118:2438-2447), an anti-inflammatory cytokine thatalleviates inflammatory arthritis (Tang et al., Science, 2011,332:478-484).

Resolving the paradox between the predicted elastase-like active sitewith the actual trypsin-like active site that is exhibited by NSP4 hasthe potential to provide structural features of the enzyme active sitethat could facilitate the development of specific NSP4 inhibitors. TheseNSP4 inhibitors may serve a need for treatment of neutrophil-mediateddiseases or disorders where the underlying pathology is completely orpartially due to the activity of NSP4.

All references cited herein, including patent applications, patentpublications, scientific literature, and National Center forBiotechnology Information (NCBI) Accession numbers are hereinincorporated by reference in their entirety, as if each individualreference were specifically and individually indicated to beincorporated by reference.

BRIEF SUMMARY

The invention broadly provides neutrophil serine protease 4 (NSP4)inhibitors and methods of using the same.

In one aspect, provided herein is a method for treating or preventing adisease or disorder mediated by granulocytes in an individual comprisingadministering to the individual an effective amount of an NSP4inhibitor. In some embodiments, the disease or disorder is aneosinophil-mediated, basophil-mediated, or a neutrophil-mediated diseaseor disorder. In some embodiments of the methods described herein, thedisease or disorder that can be treated by a NSP4 inhibitor is avascular disease, an inflammatory disease or an autoimmune disease. Insome embodiments of the methods described herein, the disease ordisorder is selected from the group consisting of stroke, diabeticretinopathy, edema, diabetic macular edema, hereditary angioedema,idiopathic angioedema, leakage of vasculature, cerebral ischemia, acutelung injury, anaphylaxis, systemic anaphylaxis, allergic lunginflammation, asthma (e.g., allergic asthma, virus-induced asthma),chronic obstructive pulmonary disease (COPD), acute respiratory distresssyndrome (ARDS), idiopathic pulmonary fibrosis, systemic lupuserythematosus (SLE), autoimmune vasculitides, blistering skin diseases(e.g., bullous pemphigoid), inflammatory skin diseases (e.g., atopicdermatitis, urticarial, eosinophilic cellulitis), cancer (e.g., lungcancer), kidney diseases (e.g., glomerulonephritis), osteoarthritis,rheumatoid arthritis, psoriatic arthritis, psoriasis, septic shock,inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease).In some embodiments of the methods described herein, the individual hasthe disease or disorder or has been diagnosed with the disease ordisorder. In some embodiments of the methods described herein, theindividual is at risk of developing the disease or disorder. In someembodiments, the individual is a human.

In one aspect, provided herein is a method for treating or preventing aneutrophil-mediated disease or disorder in an individual comprisingadministering to the individual an effective amount of an NSP4inhibitor. In some embodiments, the neutrophil-mediated disease ordisorder is selected from the group consisting of vascular disease andinflammatory disease. In a further embodiment, the vascular disease isselected from the group consisting of stroke, diabetic retinopathy,edema, diabetic macular edema, hereditary angioedema, idiopathicangioedema, leakage of vasculature, and cerebral ischemia. In anotherfurther embodiment, the inflammatory disease is selected from the groupconsisting of acute lung injury, asthma, chronic obstructive pulmonarydisease (COPD), acute respiratory distress syndrome (ARDS),osteoarthritis, rheumatoid arthritis, and septic shock. In any of theembodiments herein, the individual can be a human.

In another aspect, provided herein is an NSP4 inhibitor that can be usedin any of the methods described herein. In some embodiments, the NSP4inhibitor is selected from the group consisting of an antibody, anantisense molecule, a siRNA, a small molecule inhibitor, a proteaseinhibitor, and a peptide inhibitor. In a further embodiment, theprotease inhibitor is a serine protease inhibitor. In another furtherembodiment, the protease inhibitor is α1-antitrypsin, heparin-activatedantithrombin, C1 inhibitor, or α2-antiplasmin.

In any of the embodiments herein, the NSP4 inhibitor can be an anti-NSP4antibody that specifically binds to a NSP4. In any of the embodimentsherein, the NSP4 inhibitor can be an anti-NSP4 antibody thatspecifically binds to a mature form of NSP4. In any of the embodimentsherein, the NSP4 inhibitor can be an anti-NSP4 antibody thatspecifically binds to a mature form of NSP4 but does not bind to aprecursor form of NSP4. In any of the embodiments herein, the NSP4inhibitor can be an anti-NSP4 antibody that specifically binds to ahuman NSP4. In any of the embodiments herein, the NSP4 inhibitor can bean anti-NSP4 antibody that specifically binds to a mouse NSP4. In any ofthe embodiments herein, the NSP4 inhibitor can be an anti-NSP4 antibodythat specifically binds to both a human NSP4 and a mouse NSP4. In any ofthe embodiments herein, the anti-NSP4 antibody can be a monoclonalantibody. In any of the embodiments herein, the anti-NSP4 antibody canbe an antibody fragment selected from the group consisting of Fab,Fab′-SH, Fv, scFv, and (Fab′)₂ fragments. In any of the embodimentsherein, the anti-NSP4 antibody can be a humanized antibody or a chimericantibody. In any of the embodiments herein, the anti-NSP4 antibody cancomprise at least one, two, three, four, five, or six HVRs selected from(i) HVR-L1 comprising the amino acid sequence of SEQ ID NO:10; (ii)HVR-L2 comprising the amino acid sequence of SEQ ID NO:11; (iii) HVR-L3comprising the amino acid sequence of SEQ ID NO:12; (iv) HVR-H1comprising the amino acid sequence of SEQ ID NO:1, 4, or 7; (v) HVR-H2comprising the amino acid sequence of SEQ ID NO:2, 5, or 8; (vi) HVR-H3comprising the amino acid sequence of SEQ ID NO:3, 6, or 9. In any ofthe embodiments herein, the anti-NSP4 antibody can comprise a lightchain variable region comprising the amino acid sequence of SEQ IDNO:16, and a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:13, 14 or 15.

In some of the embodiments herein, the NSP4 inhibitor can beadministered intravenously, intramuscularly, subcutaneously, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally. In any of the embodiments herein, the NSP4 inhibitor canbe formulated in a pharmaceutical composition comprising the NSP4inhibitor and a pharmaceutically acceptable carrier.

In yet another aspect, provided herein is an article of manufacturecomprising a NSP4 inhibitor and a package insert comprising instructionsfor using the NSP4 inhibitor to treat or prevent a disease or disordermediated by granulocytes in an individual. In some embodiments, thedisease or disorder is an eosinophil-mediated, basophil-mediated, or aneutrophil-mediated disease or disorder. In some embodiments, thedisease or disorder that can be treated by a NSP4 inhibitor is avascular disease, an inflammatory disease, or an autoimmune disease. Insome embodiments, the disease or disorder is selected from the groupconsisting of stroke, diabetic retinopathy, edema, diabetic macularedema, hereditary angioedema, idiopathic angioedema, leakage ofvasculature, cerebral ischemia, acute lung injury, anaphylaxis, systemicanaphylaxis, allergic lung inflammation, asthma (e.g., allergic asthma,virus-induced asthma), chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), idiopathic pulmonaryfibrosis, systemic lupus erythematosus (SLE), autoimmune vasculitides,blistering skin diseases (e.g., bullous pemphigoid), inflammatory skindiseases (e.g., atopic dermatitis, urticarial, eosinophilic cellulitis),cancer (e.g., lung cancer), kidney diseases (e.g., glomerulonephritis),osteoarthritis, rheumatoid arthritis, psoriatic arthritis, psoriasis,septic shock, inflammatory bowel disease (e.g., ulcerative colitis,Crohn's disease). In some embodiments, the individual has the disease ordisorder or has been diagnosed with the disease or disorder. In someembodiments, the individual is at risk of developing the disease ordisorder. In some embodiments, the individual is a human.

In yet another aspect, provided herein is an article of manufacturecomprising a NSP4 inhibitor and a package insert comprising instructionsfor using the NSP4 inhibitor to treat or prevent a neutrophil-mediateddisease or disorder in an individual. In some embodiments, theneutrophil-mediated disease or disorder is selected from the groupconsisting of vascular disease and inflammatory disease. In a furtherembodiment, the vascular disease is selected from the group consistingof stroke, diabetic retinopathy, edema, diabetic macular edema,hereditary angioedema, idiopathic angioedema, leakage of vasculature,and cerebral ischemia. In another further embodiment, the inflammatorydisease is selected from the group consisting of acute lung injury,asthma, chronic obstructive pulmonary disease (COPD), acute respiratorydistress syndrome (ARDS), osteoarthritis, rheumatoid arthritis, andseptic shock. In any of the embodiments herein, the individual can be ahuman.

In another aspect, provided here is an anti-NSP4 antibody comprising atleast one, two, three, four, five or six hypervariable region (HVR)sequences selected from the group consisting of: (a) HVR-H1 comprisingthe sequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3 comprising thesequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSX₁X₂X₃PX₄T (SEQ ID NO:95), wherein X₁ is Y or A; X₂ is T, G, or D; X₃is T or F; and X₄ is P or L. In yet another aspect, provided herein isan anti-NSP4 antibody comprising a heavy chain and a light chain,wherein (a) the heavy chain comprises an HVR-H1 comprising the sequenceof GFTFSDNDIS (SEQ ID NO:50), an HVR-H2 comprising the sequence ofGSISPDNGDTNYADSVKG (SEQ ID NO:51), and an HVR-H3 comprising the sequenceof RDDVPAVFTSAMDY (SEQ ID NO:52); and/or (b) the light chain comprisesan HVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2comprising the sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3comprising the sequence of QQSX₁X₂X₃PX₄T (SEQ ID NO:95), wherein X₁ is Yor A; X₂ is T, G, or D; X₃ is T or F; and X₄ is P or L. In certainembodiments, the HVR-L3 comprises the sequence selected from the groupconsisting of SEQ ID NO:12 and 92-94.

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3 comprising thesequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the anti-NSP4 antibodycomprises a heavy chain and a light chain, wherein (a) the heavy chaincomprises an HVR-H1 comprising the sequence of GFTFSDNDIS (SEQ IDNO:50), an HVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG (SEQ IDNO:51), and an HVR-H3 comprising the sequence of RDDVPAVFTSAMDY (SEQ IDNO:52); and/or (b) the light chain comprises an HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence ofSASFLYS (SEQ ID NO:11), and an HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprisesa heavy chain variable region comprising the sequence of SEQ ID NO:78and/or a light chain variable region comprising the sequence of SEQ IDNO:16. In certain embodiments, the antibody comprises a variable domaincomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences of antibody 35.WT (e.g., as shown in Tables 5-7).In certain embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody 35.WT (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3 comprising thesequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYGFPLT (SEQ ID NO:92). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSDNDIS (SEQ ID NO:50), anHVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51), andan HVR-H3 comprising the sequence of RDDVPAVFTSAMDY (SEQ ID NO:52);and/or (b) the light chain comprises an HVR-L1 comprising the sequenceof RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYGFPLT (SEQID NO:92). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:78 and/or a lightchain variable region comprising the sequence of SEQ ID NO:102. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 35.14(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 35.14 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3 comprising thesequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYDFPLT (SEQ ID NO:93). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSDNDIS (SEQ ID NO:50), anHVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51), andan HVR-H3 comprising the sequence of RDDVPAVFTSAMDY (SEQ ID NO:52);and/or (b) the light chain comprises an HVR-L1 comprising the sequenceof RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYDFPLT (SEQID NO:93). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:78 and/or a lightchain variable region comprising the sequence of SEQ ID NO:103. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 35.50(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 35.50 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3 comprising thesequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSAGFPLT (SEQ ID NO:94). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSDNDIS (SEQ ID NO:50), anHVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51), andan HVR-H3 comprising the sequence of RDDVPAVFTSAMDY (SEQ ID NO:52);and/or (b) the light chain comprises an HVR-L1 comprising the sequenceof RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSAGFPLT (SEQID NO:94). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:78 and/or a lightchain variable region comprising the sequence of SEQ ID NO:104. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 35.62(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 35.62 (e.g., as shown in Table 7).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising (a) an HVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG(SEQ ID NO:51); (b) an HVR-H3 comprising the sequence of RDDVPAVFTSAMDY(SEQ ID NO:52); and (c) an HVR-L3 comprising the sequence ofQQSX₁X₂X₃PX₄T (SEQ ID NO:95), wherein X₁ is Y or A; X₂ is T, G, or D; X₃is T or F; and X₄ is P or L. In certain embodiments, the antibodycomprises (a) an HVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG(SEQ ID NO:51); (b) an HVR-H3 comprising the sequence of RDDVPAVFTSAMDY(SEQ ID NO:52); and (c) an HVR-L3 comprising the sequence of QQSYTTPPT(SEQ ID NO:12). In certain embodiments, the antibody comprises (a) anHVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (b)an HVR-H3 comprising the sequence of RDDVPAVFTSAMDY (SEQ ID NO:52); and(c) an HVR-L3 comprising the sequence of QQSYGFPLT (SEQ ID NO:92). Incertain embodiments, the antibody comprises (a) an HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (b) an HVR-H3 comprisingthe sequence of RDDVPAVFTSAMDY (SEQ ID NO:52); and (c) an HVR-L3comprising the sequence of QQSYDFPLT (SEQ ID NO:93). In certainembodiments, the antibody comprises (a) an HVR-H2 comprising thesequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (b) an HVR-H3 comprisingthe sequence of RDDVPAVFTSAMDY (SEQ ID NO:52); and (c) an HVR-L3comprising the sequence of QQSAGFPLT (SEQ ID NO:94).

In another aspect, provided herein is an anti-NSP4 antibody comprisingat least one, two, three, four, five or six hypervariable region (HVR)sequences selected from the group consisting of: (a) HVR-H1 comprisingthe sequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising thesequence of KRHLHNVAFDY (SEQ ID NO:87); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQAYSAPPT (SEQ ID NO:96). In some embodiments, the antibody comprises aheavy chain and a light chain, wherein (a) the heavy chain comprises anHVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), an HVR-H2comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66), and anHVR-H3 comprising the sequence of KRHLHNVAFDY (SEQ ID NO:87); and/or (b)the light chain comprises an HVR-L1 comprising the sequence of RASQDVS(SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS (SEQ IDNO:11), and an HVR-L3 comprising the sequence of QQAYSAPPT (SEQ IDNO:96). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:105 and/or a lightchain variable region comprising the sequence of SEQ ID NO:106. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 35.77(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 35.77 (e.g., as shown in Table 7).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising (a) an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG(SEQ ID NO:66); (b) an HVR-H3 comprising the sequence of KRHLHNVAFDY(SEQ ID NO:87); and (c) an HVR-L3 comprising the sequence of QQAYSAPPT(SEQ ID NO:96).

In another aspect, provided herein an anti-NSP4 antibody comprising atleast one, two, three, four, five or six hypervariable region (HVR)sequences selected from the group consisting of: (a) HVR-H1 comprisingthe sequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66) or the sequence ofAWIPTAGGNTYYADSVKG (SEQ ID NO:88); (c) HVR-H3 comprising the sequence ofX₁X₂X₃FHNVAFDY (SEQ ID NO:91), wherein X₁ is K or R; X₂ is S, G, or V;and X₃ is L or F; (d) HVR-L1 comprising the sequence of RASQDVS (SEQ IDNO:19); (e) HVR-L2 comprising the sequence of SASFLYS (SEQ ID NO:11);and (f) HVR-L3 comprising the sequence of QQX₁X₂X₃X₄PPT (SEQ ID NO:101),wherein X₁ is S, A, N, or T; X₂ is Y, N, or F; X₃ is T, S, or N; and X₄is T, A, or S. In certain embodiments, the antibody comprises a heavychain and a light chain, wherein (a) the heavy chain comprises an HVR-H1comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), an HVR-H2comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66) or thesequence of AWIPTAGGNTYYADSVKG (SEQ ID NO:88), and an HVR-H3 comprisingthe sequence of X₁X₂X₃FHNVAFDY (SEQ ID NO:91), wherein X₁ is K or R; X₂is S, G, or V; and X₃ is L or F; and/or (b) the light chain comprises anHVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2comprising the sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3comprising the sequence of QQX₁X₂X₃X₄PPT (SEQ ID NO:101), wherein X₁ isS, A, N, or T; X₂ is Y, N, or F; X₃ is T, S, or N; and X₄ is T, A, or S.In certain embodiments, the HVR-H3 comprises the sequence selected fromthe group consisting of SEQ ID NO:67, 89, and 90. In certainembodiments, the HVR-L3 comprises the sequence selected from the groupconsisting of SEQ ID NO:12 and 97-100. In certain embodiments, theHVR-H3 comprises the sequence selected from the group consisting of SEQID NO:67, 89, and 90, and the HVR-L3 comprises the sequence selectedfrom the group consisting of SEQ ID NO:12 and 97-100.

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising thesequence of KSLFHNVAFDY (SEQ ID NO:67); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), anHVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66), andan HVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); and/or(b) the light chain comprises an HVR-L1 comprising the sequence ofRASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYTTPPT (SEQID NO:12). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:83 and/or a lightchain variable region comprising the sequence of SEQ ID NO:16. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 51.WT(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.WT (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWIPTAGGNTYYADSVKG (SEQ ID NO:88); (c) HVR-H3 comprising thesequence of KSLFHNVAFDY (SEQ ID NO:67); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTAPPT (SEQ ID NO:97). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), anHVR-H2 comprising the sequence of AWIPTAGGNTYYADSVKG (SEQ ID NO:88), andan HVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); and/or(b) the light chain comprises an HVR-L1 comprising the sequence ofRASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYTAPPT (SEQID NO:97). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:107 and/or a lightchain variable region comprising the sequence of SEQ ID NO:108. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 51.30(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.30 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises a variabledomain comprising at least one, two, three, four, five or sixhypervariable region (HVR) sequences selected from the group consistingof: (a) HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65); (b)HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c)HVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQANSTPPT (SEQ ID NO:98). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSGSGIH(SEQ ID NO:65), an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG(SEQ ID NO:66), and an HVR-H3 comprising the sequence of KSLFHNVAFDY(SEQ ID NO:67); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQANSTPPT (SEQ ID NO:98). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:83 and/or a light chain variable region comprising thesequence of SEQ ID NO:109. In certain embodiments, the antibodycomprises a variable domain comprising at least one, two, three, four,five or six hypervariable region (HVR) sequences of antibody 51.50(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.50 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising thesequence of RGLFHNVAFDY (SEQ ID NO:89); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTAPPT (SEQ ID NO:97). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), anHVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66), andan HVR-H3 comprising the sequence of RGLFHNVAFDY (SEQ ID NO:89); and/or(b) the light chain comprises an HVR-L1 comprising the sequence ofRASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYTAPPT (SEQID NO:97). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:110 and/or a lightchain variable region comprising the sequence of SEQ ID NO:108. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 51.51(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.51 (e.g., as shown in Table 7).

In certain embodiments, the NSP4 antibody comprises at least one, two,three, four, five or six hypervariable region (HVR) sequences selectedfrom the group consisting of: (a) HVR-H1 comprising the sequence ofGFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising the sequence ofAWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising the sequence ofRVFFHNVAFDY (SEQ ID NO:90); (d) HVR-L1 comprising the sequence ofRASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11); and (f) HVR-L3 comprising the sequence of QQNFSSPPT (SEQID NO:99). In certain embodiments, the antibody comprises a heavy chainand a light chain, wherein (a) the heavy chain comprises an HVR-H1comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), an HVR-H2comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66), and anHVR-H3 comprising the sequence of RVFFHNVAFDY (SEQ ID NO:90); and/or (b)the light chain comprises an HVR-L1 comprising the sequence of RASQDVS(SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS (SEQ IDNO:11), and an HVR-L3 comprising the sequence of QQNFSSPPT (SEQ IDNO:99). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:111 and/or a lightchain variable region comprising the sequence of SEQ ID NO:112. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 51.59(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.59 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising thesequence of KSLFHNVAFDY (SEQ ID NO:67); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTAPPT (SEQ ID NO:97). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ ID NO:65), anHVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66), andan HVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); and/or(b) the light chain comprises an HVR-L1 comprising the sequence ofRASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYTAPPT (SEQID NO:97). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:83 and/or a lightchain variable region comprising the sequence of SEQ ID NO:108. Incertain embodiments, the antibody comprises at least one, two, three,four, five or six hypervariable region (HVR) sequences of antibody 51.72(e.g., as shown in Tables 5-7). In certain embodiments, the antibodycomprises a heavy chain variable region sequence and/or a light chainvariable region sequence of antibody 51.72 (e.g., as shown in Table 7).

In certain embodiments, the anti-NSP4 antibody comprises at least one,two, three, four, five or six hypervariable region (HVR) sequencesselected from the group consisting of: (a) HVR-H1 comprising thesequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3 comprising thesequence of RGLFHNVAFDY (SEQ ID NO:89); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQTYNAPPT (SEQ ID NO:100). In certain embodiments, the antibodycomprises a heavy chain and a light chain, wherein (a) the heavy chaincomprises an HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ IDNO:65), an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ IDNO:66), and an HVR-H3 comprising the sequence of RGLFHNVAFDY (SEQ IDNO:89); and/or (b) the light chain comprises an HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence ofSASFLYS (SEQ ID NO:11), and an HVR-L3 comprising the sequence ofQQTYNAPPT (SEQ ID NO:100). In certain embodiments, the antibodycomprises a heavy chain variable region comprising the sequence of SEQID NO:110 and/or a light chain variable region comprising the sequenceof SEQ ID NO:113. In certain embodiments, the antibody comprises atleast one, two, three, four, five or six hypervariable region (HVR)sequences of antibody 51.82 (e.g., as shown in Tables 5-7). In certainembodiments, the antibody comprises a heavy chain variable regionsequence and/or a light chain variable region sequence of antibody 51.82(e.g., as shown in Table 7).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising (a) an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG(SEQ ID NO:66) or the sequence of AWIPTAGGNTYYADSVKG (SEQ ID NO:88); (b)an HVR-H3 comprising the sequence of X₁X₂X₃FHNVAFDY (SEQ ID NO:91),wherein X₁ is K or R; X₂ is S, G, or V; and X₃ is L or F; and (c) anHVR-L3 comprising the sequence of QQX₁X₂X₃X₄PPT (SEQ ID NO:101), whereinX₁ is S, A, N, or T; X₂ is Y, N, or F; X₃ is T, S, or N; and X₄ is T, A,or S. In certain embodiments, the antibody comprises (a) an HVR-H2comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (b) anHVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); and (c) anHVR-L3 comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises (a) an HVR-H2 comprising thesequence of AWIPTAGGNTYYADSVKG (SEQ ID NO:88); (b) an HVR-H3 comprisingthe sequence of KSLFHNVAFDY (SEQ ID NO:67); and (c) an HVR-L3 comprisingthe sequence of QQSYTAPPT (SEQ ID NO:97). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAWISPTGGNTYYADSVKG (SEQ ID NO:66); (b) an HVR-H3 comprising the sequenceof KSLFHNVAFDY (SEQ ID NO:67); and (c) an HVR-L3 comprising the sequenceof QQANSTPPT (SEQ ID NO:98). In certain embodiments, the antibodycomprises (a) an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG(SEQ ID NO:66); (b) an HVR-H3 comprising the sequence of RGLFHNVAFDY(SEQ ID NO:89); and (c) an HVR-L3 comprising the sequence of QQSYTAPPT(SEQ ID NO:97). In certain embodiments, the antibody comprises (a) anHVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (b)an HVR-H3 comprising the sequence of RVFFHNVAFDY (SEQ ID NO:90); and (c)an HVR-L3 comprising the sequence of QQNFSSPPT (SEQ ID NO:99). Incertain embodiments, the antibody comprises (a) an HVR-H2 comprising thesequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (b) an HVR-H3 comprisingthe sequence of KSLFHNVAFDY (SEQ ID NO:67); and (c) an HVR-L3 comprisingthe sequence of QQSYTAPPT (SEQ ID NO:97). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAWISPTGGNTYYADSVKG (SEQ ID NO:66); (b) an HVR-H3 comprising the sequenceof RGLFHNVAFDY (SEQ ID NO:89); and (c) an HVR-L3 comprising the sequenceof QQTYNAPPT (SEQ ID NO:100).

In another aspect, provided herein is an anti-NSP4 antibody comprisingat least one, two, three, four, five or six hypervariable region (HVR)sequences selected from the group consisting of: (a) HVR-H1 comprisingthe sequence of GFTFSGSWIS (SEQ ID NO:20); (b) HVR-H2 comprising thesequence of GTISPYNGSTYYADSVKG (SEQ ID NO:21); (c) HVR-H3 comprising thesequence of RVLRPKVYASVMDY (SEQ ID NO:22); (d) HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprising the sequenceof SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprises(a) HVR-H2 comprising the sequence of GTISPYNGSTYYADSVKG (SEQ ID NO:21);(b) HVR-H3 comprising the sequence of RVLRPKVYASVMDY (SEQ ID NO:22); and(c) an HVR-L3 comprising the sequence of QQSYTTPPT (SEQ ID NO:12). Incertain embodiments, the antibody comprises a heavy chain and a lightchain, wherein (a) the heavy chain comprises an HVR-H1 comprising thesequence of GFTFSGSWIS (SEQ ID NO:20), an HVR-H2 comprising the sequenceof GTISPYNGSTYYADSVKG (SEQ ID NO:21), and an HVR-H3 comprising thesequence of RVLRPKVYASVMDY (SEQ ID NO:22); and/or (b) the light chaincomprises an HVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19),an HVR-L2 comprising the sequence of SASFLYS (SEQ ID NO:11), and anHVR-L3 comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the sequence of SEQ ID NO:68 and/or a light chain variableregion comprising the sequence of SEQ ID NO:16. In certain embodiments,the antibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody 1-1 (e.g., as shown inTables 3 and 4 and SEQ ID NO:19, 11, and 12). In certain embodiments,the antibody comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody 1-1 (e.g., as shown inTable 4 and SEQ ID NO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSGYSIH (SEQ ID NO:23); (b) HVR-H2comprising the sequence of AGISPTNGYTDYADSVKG (SEQ ID NO:24); (c) HVR-H3comprising the sequence of RLVFYRGVMDY (SEQ ID NO:25); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAGISPTNGYTDYADSVKG (SEQ ID NO:24); (b) an HVR-H3 comprising the sequenceof RLVFYRGVMDY (SEQ ID NO:25); and (c) an HVR-L3 comprising the sequenceof QQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibodycomprises a heavy chain and a light chain, wherein (a) the heavy chaincomprises an HVR-H1 comprising the sequence of GFTFSGYSIH (SEQ IDNO:23), an HVR-H2 comprising the sequence of AGISPTNGYTDYADSVKG (SEQ IDNO:24), and an HVR-H3 comprising the sequence of RLVFYRGVMDY (SEQ IDNO:25); and/or (b) the light chain comprises an HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence ofSASFLYS (SEQ ID NO:11), and an HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprisesa heavy chain variable region comprising the sequence of SEQ ID NO:69and/or a light chain variable region comprising the sequence of SEQ IDNO:16. In certain embodiments, the antibody comprises at least one, two,three, four, five, or six hypervariable region (HVR) sequences ofantibody 1-2 (e.g., as shown in Tables 3 and 4 and SEQ ID NO:19, 11, and12). In certain embodiments, the antibody comprises a heavy chainvariable region sequence and/or a light chain variable region sequenceof antibody 1-2 (e.g., as shown in Table 4 and SEQ ID NO:19, 11, and12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSDNWIS (SEQ ID NO:26); (b) HVR-H2comprising the sequence of GYIYPASGYTDYADSVKG (SEQ ID NO:27); (c) HVR-H3comprising the sequence of SDSPHAYWYAMDY (SEQ ID NO:28); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGYIYPASGYTDYADSVKG (SEQ ID NO:27); (b) an HVR-H3 comprising the sequenceof SDSPHAYWYAMDY (SEQ ID NO:28); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSDNWIS(SEQ ID NO:26), an HVR-H2 comprising the sequence of GYIYPASGYTDYADSVKG(SEQ ID NO:27), and an HVR-H3 comprising the sequence of SDSPHAYWYAMDY(SEQ ID NO:28); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:70 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 1-3 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 1-3 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTNNSIS (SEQ ID NO:29); (b) HVR-H2comprising the sequence of GAISPNNGSTYYADSVKG (SEQ ID NO:30); (c) HVR-H3comprising the sequence of RNAWHYSWVGVMDY (SEQ ID NO:31); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGAISPNNGSTYYADSVKG (SEQ ID NO:30); (b) an HVR-H3 comprising the sequenceof RNAWHYSWVGVMDY (SEQ ID NO:31); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFTNNSIS(SEQ ID NO:29), an HVR-H2 comprising the sequence of GAISPNNGSTYYADSVKG(SEQ ID NO:30), and an HVR-H3 comprising the sequence of RNAWHYSWVGVMDY(SEQ ID NO:31); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:71 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 1-5 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 1-5 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTDYSIH (SEQ ID NO:32); (b) HVR-H2comprising the sequence of AEIYPYSGDTYYADSVKG (SEQ ID NO:33); (c) HVR-H3comprising the sequence of RDGDGWFDWAMDY (SEQ ID NO:34); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAEIYPYSGDTYYADSVKG (SEQ ID NO:33); (b) an HVR-H3 comprising the sequenceof RDGDGWFDWAMDY (SEQ ID NO:34); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFTDYSIH(SEQ ID NO:32), an HVR-H2 comprising the sequence of AEIYPYSGDTYYADSVKG(SEQ ID NO:33), and an HVR-H3 comprising the sequence of RDGDGWFDWAMDY(SEQ ID NO:34); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:72 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 2-1 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 2-1 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSSTAIS (SEQ ID NO:35); (b) HVR-H2comprising the sequence of GEIYPSDGYTDYADSVKG (SEQ ID NO:36); (c) HVR-H3comprising the sequence of RVKWAVSSLGVMDY (SEQ ID NO:37); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGEIYPSDGYTDYADSVKG (SEQ ID NO:36); (b) an HVR-H3 comprising the sequenceof RVKWAVSSLGVMDY (SEQ ID NO:37); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSSTAIS(SEQ ID NO:35), an HVR-H2 comprising the sequence of GEIYPSDGYTDYADSVKG(SEQ ID NO:36), and an HVR-H3 comprising the sequence of RVKWAVSSLGVMDY(SEQ ID NO:37); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:73 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 2-2 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 2-2 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTDSDIS (SEQ ID NO:38); (b) HVR-H2comprising the sequence of AWISPSDGATDYADSVKG (SEQ ID NO:39); (c) HVR-H3comprising the sequence of HEASDDDYAIDY (SEQ ID NO:40); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAWISPSDGATDYADSVKG (SEQ ID NO:39); (b) an HVR-H3 comprising the sequenceof HEASDDDYAIDY (SEQ ID NO:40); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFTDSDIS(SEQ ID NO:38), an HVR-H2 comprising the sequence of AWISPSDGATDYADSVKG(SEQ ID NO:39), and an HVR-H3 comprising the sequence of HEASDDDYAIDY(SEQ ID NO:40); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:74 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 2-3 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 2-3 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSDYWIS (SEQ ID NO:41); (b) HVR-H2comprising the sequence of AGISPNNGDTYYADSVKG (SEQ ID NO:42); (c) HVR-H3comprising the sequence of REDDDERDYAMDY (SEQ ID NO:43); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAGISPNNGDTYYADSVKG (SEQ ID NO:42); (b) an HVR-H3 comprising the sequenceof REDDDERDYAMDY (SEQ ID NO:43); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSDYWIS(SEQ ID NO:41), an HVR-H2 comprising the sequence of AGISPNNGDTYYADSVKG(SEQ ID NO:42), and an HVR-H3 comprising the sequence of REDDDERDYAMDY(SEQ ID NO:43); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:75 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 2-4 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 2-4 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTGYGIS (SEQ ID NO:44); (b) HVR-H2comprising the sequence of GWIYPASGATYYADSVKG (SEQ ID NO:45); (c) HVR-H3comprising the sequence of RHRAFDWYPYYIGSSVMDY (SEQ ID NO:46); (d)HVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2comprising the sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises (a) an HVR-H2 comprising thesequence of GWIYPASGATYYADSVKG (SEQ ID NO:45); (b) an HVR-H3 comprisingthe sequence of RHRAFDWYPYYIGSSVMDY (SEQ ID NO:46); and (c) an HVR-L3comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises a heavy chain and a light chain,wherein (a) the heavy chain comprises an HVR-H1 comprising the sequenceof GFTFTGYGIS (SEQ ID NO:44), an HVR-H2 comprising the sequence ofGWIYPASGATYYADSVKG (SEQ ID NO:45), and an HVR-H3 comprising the sequenceof RHRAFDWYPYYIGSSVMDY (SEQ ID NO:46); and/or (b) the light chaincomprises an HVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19),an HVR-L2 comprising the sequence of SASFLYS (SEQ ID NO:11), and anHVR-L3 comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the sequence of SEQ ID NO:76 and/or a light chain variableregion comprising the sequence of SEQ ID NO:16. In certain embodiments,the antibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody 2-5 (e.g., as shown inTables 3 and 4 and SEQ ID NO:19, 11, and 12). In certain embodiments,the antibody comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody 2-5 (e.g., as shown inTable 4 and SEQ ID NO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSDYSIS (SEQ ID NO:47); (b) HVR-H2comprising the sequence of GEINPAGGATYYADSVKG (SEQ ID NO:48); (c) HVR-H3comprising the sequence of RGDFPFWSDAYYVMDY (SEQ ID NO:49); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGEINPAGGATYYADSVKG (SEQ ID NO:48); (b) an HVR-H3 comprising the sequenceof RGDFPFWSDAYYVMDY (SEQ ID NO:49); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSDYSIS(SEQ ID NO:47), an HVR-H2 comprising the sequence of GEINPAGGATYYADSVKG(SEQ ID NO:48), and an HVR-H3 comprising the sequence ofRGDFPFWSDAYYVMDY (SEQ ID NO:49); and/or (b) the light chain comprises anHVR-L1 comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2comprising the sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3comprising the sequence of QQSYTTPPT (SEQ ID NO:12). In certainembodiments, the antibody comprises a heavy chain variable regioncomprising the sequence of SEQ ID NO:77 and/or a light chain variableregion comprising the sequence of SEQ ID NO:16. In certain embodiments,the antibody comprises at least one, two, three, four, five, or sixhypervariable region (HVR) sequences of antibody 3-2 (e.g., as shown inTables 3 and 4 and SEQ ID NO:19, 11, and 12). In certain embodiments,the antibody comprises a heavy chain variable region sequence and/or alight chain variable region sequence of antibody 3-2 (e.g., as shown inTable 4 and SEQ ID NO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSDNDIS (SEQ ID NO:50); (b) HVR-H2comprising the sequence of GSISPDNGDTNYADSVKG (SEQ ID NO:51); (c) HVR-H3comprising the sequence of RDDVPAVFTSAMDY (SEQ ID NO:52); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGSISPDNGDTNYADSVKG (SEQ ID NO:51); (b) an HVR-H3 comprising the sequenceof RDDVPAVFTSAMDY (SEQ ID NO:52); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSDNDIS(SEQ ID NO:50), an HVR-H2 comprising the sequence of GSISPDNGDTNYADSVKG(SEQ ID NO:51), and an HVR-H3 comprising the sequence of RDDVPAVFTSAMDY(SEQ ID NO:52); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:78 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. the antibody comprises at least one, two,three, four, five, or six hypervariable region (HVR) sequences ofantibody 3-5 (e.g., as shown in Tables 3 and 4 and SEQ ID NO:19, 11, and12). In certain embodiments, the antibody comprises a heavy chainvariable region sequence and/or a light chain variable region sequenceof antibody 3-5 (e.g., as shown in Table 4 and SEQ ID NO:19, 11, and12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSGSDIS (SEQ ID NO:53); (b) HVR-H2comprising the sequence of GEIYPSNGDTYYADSVKG (SEQ ID NO:54); (c) HVR-H3comprising the sequence of RSVRPSWWAMDY (SEQ ID NO:55); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGEIYPSNGDTYYADSVKG (SEQ ID NO:54); (b) an HVR-H3 comprising the sequenceof RSVRPSWWAMDY (SEQ ID NO:55); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSGSDIS(SEQ ID NO:53), an HVR-H2 comprising the sequence of GEIYPSNGDTYYADSVKG(SEQ ID NO:54), and an HVR-H3 comprising the sequence of RSVRPSWWAMDY(SEQ ID NO:55); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:79 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 4-2 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 4-2 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSSYDIS (SEQ ID NO:56); (b) HVR-H2comprising the sequence of GTISPYDGYTDYADSVKG (SEQ ID NO:57); (c) HVR-H3comprising the sequence of RYIRRYSVHYGMDY (SEQ ID NO:58); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGTISPYDGYTDYADSVKG (SEQ ID NO:57); (b) an HVR-H3 comprising the sequenceof RYIRRYSVHYGMDY (SEQ ID NO:58); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSSYDIS(SEQ ID NO:56), an HVR-H2 comprising the sequence of GTISPYDGYTDYADSVKG(SEQ ID NO:57), and an HVR-H3 comprising the sequence of RYIRRYSVHYGMDY(SEQ ID NO:58); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:80 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 4-3 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 4-3 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTSTSIH (SEQ ID NO:59); (b) HVR-H2comprising the sequence of AEITPHGGYTNYADSVKG (SEQ ID NO:60); (c) HVR-H3comprising the sequence of RGRTKWGWLYGMDY (SEQ ID NO:61); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAEITPHGGYTNYADSVKG (SEQ ID NO:60); (b) an HVR-H3 comprising the sequenceof RGRTKWGWLYGMDY (SEQ ID NO:61); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFTSTSIH(SEQ ID NO:59), an HVR-H2 comprising the sequence of AEITPHGGYTNYADSVKG(SEQ ID NO:60), and an HVR-H3 comprising the sequence of RGRTKWGWLYGMDY(SEQ ID NO:61); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:81 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 4-4 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 4-4 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTNNSIH (SEQ ID NO:62); (b) HVR-H2comprising the sequence of AEIAPDDGYTYYADSVKG (SEQ ID NO:63); (c) HVR-H3comprising the sequence of RGVIRYAYLYAMDY (SEQ ID NO:64); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAEIAPDDGYTYYADSVKG (SEQ ID NO:63); (b) an HVR-H3 comprising the sequenceof RGVIRYAYLYAMDY (SEQ ID NO:64); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFTNNSIH(SEQ ID NO:62), an HVR-H2 comprising the sequence of AEIAPDDGYTYYADSVKG(SEQ ID NO:63), and an HVR-H3 comprising the sequence of RGVIRYAYLYAMDY(SEQ ID NO:64); and/or (b) the light chain comprises an HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:82 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 4-5 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 4-5 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSGSGIH (SEQ ID NO:65); (b) HVR-H2comprising the sequence of AWISPTGGNTYYADSVKG (SEQ ID NO:66); (c) HVR-H3comprising the sequence of KSLFHNVAFDY (SEQ ID NO:67); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAWISPTGGNTYYADSVKG (SEQ ID NO:66); (b) an HVR-H3 comprising the sequenceof KSLFHNVAFDY (SEQ ID NO:67); and (c) an HVR-L3 comprising the sequenceof QQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibodycomprises a heavy chain and a light chain, wherein (a) the heavy chaincomprises an HVR-H1 comprising the sequence of GFTFSGSGIH (SEQ IDNO:65), an HVR-H2 comprising the sequence of AWISPTGGNTYYADSVKG (SEQ IDNO:66), and an HVR-H3 comprising the sequence of KSLFHNVAFDY (SEQ IDNO:67); and/or (b) the light chain comprises an HVR-L1 comprising thesequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence ofSASFLYS (SEQ ID NO:11), and an HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprisesa heavy chain variable region comprising the sequence of SEQ ID NO:83and/or a light chain variable region comprising the sequence of SEQ IDNO:16. In certain embodiments, the antibody comprises at least one, two,three, four, five, or six hypervariable region (HVR) sequences ofantibody 5-1 (e.g., as shown in Tables 3 and 4 and SEQ ID NO:19, 11, and12). In certain embodiments, the antibody comprises a heavy chainvariable region sequence and/or a light chain variable region sequenceof antibody 5-1 (e.g., as shown in Table 4 and SEQ ID NO:19, 11, and12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSNTYIS (SEQ ID NO:1); (b) HVR-H2comprising the sequence of GFIYPANGATYYADSVKG (SEQ ID NO:2); (c) HVR-H3comprising the sequence of RRYRLSFDY (SEQ ID NO:3); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofGFIYPANGATYYADSVKG (SEQ ID NO:2); (b) an HVR-H3 comprising the sequenceof RRYRLSFDY (SEQ ID NO:3); and (c) an HVR-L3 comprising the sequence ofQQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibody comprisesa heavy chain and a light chain, wherein (a) the heavy chain comprisesan HVR-H1 comprising the sequence of GFTFSNTYIS (SEQ ID NO:1), an HVR-H2comprising the sequence of GFIYPANGATYYADSVKG (SEQ ID NO:2), and anHVR-H3 comprising the sequence of RRYRLSFDY (SEQ ID NO:3); and/or (b)the light chain comprises an HVR-L1 comprising the sequence of RASQDVS(SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS (SEQ IDNO:11), and an HVR-L3 comprising the sequence of QQSYTTPPT (SEQ IDNO:12). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:84 and/or a lightchain variable region comprising the sequence of SEQ ID NO:16. Incertain embodiments, the antibody comprises at least one, two, three,four, five, or six hypervariable region (HVR) sequences of antibody 5-2(e.g., as shown in Tables 3 and 4 and SEQ ID NO:19, 11, and 12). Incertain embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody 5-2 (e.g., as shown in Table 4 and SEQ ID NO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFSGNDIS (SEQ ID NO:4); (b) HVR-H2comprising the sequence of AGISPYGGSTYYADSVKG (SEQ ID NO:5); (c) HVR-H3comprising the sequence of RRVSFYSRHAGMDY (SEQ ID NO:6); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAGISPYGGSTYYADSVKG (SEQ ID NO:5); (b) an HVR-H3 comprising the sequenceof RRVSFYSRHAGMDY (SEQ ID NO:6); and (c) an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain and a light chain, wherein (a) theheavy chain comprises an HVR-H1 comprising the sequence of GFTFSGNDIS(SEQ ID NO:4), an HVR-H2 comprising the sequence of AGISPYGGSTYYADSVKG(SEQ ID NO:5), and an HVR-H3 comprising the sequence of RRVSFYSRHAGMDY(SEQ ID NO:6); and/or (b) the light chain comprises an HVR-L1 comprisingthe sequence of RASQDVS (SEQ ID NO:19), an HVR-L2 comprising thesequence of SASFLYS (SEQ ID NO:11), and an HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises a heavy chain variable region comprising the sequenceof SEQ ID NO:85 and/or a light chain variable region comprising thesequence of SEQ ID NO:16. In certain embodiments, the antibody comprisesat least one, two, three, four, five, or six hypervariable region (HVR)sequences of antibody 5-3 (e.g., as shown in Tables 3 and 4 and SEQ IDNO:19, 11, and 12). In certain embodiments, the antibody comprises aheavy chain variable region sequence and/or a light chain variableregion sequence of antibody 5-3 (e.g., as shown in Table 4 and SEQ IDNO:19, 11, and 12).

In yet another aspect, provided herein is an anti-NSP4 antibodycomprising at least one, two, three, four, five or six hypervariableregion (HVR) sequences selected from the group consisting of: (a) HVR-H1comprising the sequence of GFTFTSYAIS (SEQ ID NO:7); (b) HVR-H2comprising the sequence of AGISPSNGYTNYADSVKG (SEQ ID NO:8); (c) HVR-H3comprising the sequence of RAGRWTHSDIDY (SEQ ID NO:9); (d) HVR-L1comprising the sequence of RASQDVS (SEQ ID NO:19); (e) HVR-L2 comprisingthe sequence of SASFLYS (SEQ ID NO:11); and (f) HVR-L3 comprising thesequence of QQSYTTPPT (SEQ ID NO:12). In certain embodiments, theantibody comprises (a) an HVR-H2 comprising the sequence ofAGISPSNGYTNYADSVKG (SEQ ID NO:8); (b) an HVR-H3 comprising the sequenceof RAGRWTHSDIDY (SEQ ID NO:9); and (c) an HVR-L3 comprising the sequenceof QQSYTTPPT (SEQ ID NO:12). In certain embodiments, the antibodycomprises a heavy chain and a light chain, wherein (a) the heavy chaincomprises an HVR-H1 comprising the sequence of GFTFTSYAIS (SEQ ID NO:7),an HVR-H2 comprising the sequence of AGISPSNGYTNYADSVKG (SEQ ID NO:8),and an HVR-H3 comprising the sequence of RAGRWTHSDIDY (SEQ ID NO:9);and/or (b) the light chain comprises an HVR-L1 comprising the sequenceof RASQDVS (SEQ ID NO:19), an HVR-L2 comprising the sequence of SASFLYS(SEQ ID NO:11), and an HVR-L3 comprising the sequence of QQSYTTPPT (SEQID NO:12). In certain embodiments, the antibody comprises a heavy chainvariable region comprising the sequence of SEQ ID NO:86 and/or a lightchain variable region comprising the sequence of SEQ ID NO:16. Incertain embodiments, the antibody comprises at least one, two, three,four, five, or six hypervariable region (HVR) sequences of antibody 5-4(e.g., as shown in Tables 3 and 4 and SEQ ID NO:19, 11, and 12). Incertain embodiments, the antibody comprises a heavy chain variableregion sequence and/or a light chain variable region sequence ofantibody 5-4 (e.g., as shown in Table 4 and SEQ ID NO:19, 11, and 12).

In certain embodiments that may be combined with any of the aboveembodiments, the antibody comprises a heavy chain constant regioncomprising the sequence of SEQ ID NO:114. In certain embodiments thatmay be combined with any of the above embodiments, the antibodycomprises a light chain constant region comprising the sequence of SEQID NO:115.

In yet another aspect, provided herein is an anti-NSP4 antibody thatspecifically binds an NSP4 active site. In another aspect, providedherein is an anti-NSP4 antibody that inhibits catalytic activity ofNSP4. In another aspect, provided herein is an anti-NSP4 antibody thatspecifically binds an NSP4 active site and inhibits catalytic activityof NSP4.

In yet another aspect, provided herein is an anti-NSP4 antibody thatspecifically binds an NSP4 heparin binding site. In another aspect,provided herein is an anti-NSP4 antibody that competes with heparin forbinding to NSP4. In another aspect, provided herein is an anti-NSP4antibody that specifically binds an NSP4 heparin binding site andcompetes with heparin for binding to NSP4.

In certain embodiments that may be combined with any of the aboveembodiments, the antibody is a monoclonal antibody. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is an antibody fragment selected from the group consistingof a Fab, Fab′-SH, Fv, scFv, and (Fab′)₂ fragment. In certainembodiments that may be combined with any of the preceding embodiments,the antibody comprises a constant region of human IgG1, IgG2, IgG3, orIgG4. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody specifically binds to a mature formof a NSP4. In certain embodiments that may be combined with any of thepreceding embodiments, the antibody specifically binds to a mature formof NSP4 but does not bind to a precursor form of NSP4. In certainembodiments that may be combined with any of the preceding embodiments,the antibody specifically binds to a human NSP4. In certain embodimentsthat may be combined with any of the preceding embodiments, the antibodyspecifically binds to a mouse NSP4. In certain embodiments that may becombined with any of the preceding embodiments, the antibodyspecifically binds to both a human NSP4 and a mouse NSP4.

In another aspect, provided herein is an isolated nucleic acid encodingany of the antibodies described herein. In another aspect, providedherein is a vector comprising the nucleic acid described herein. Inanother aspect, provided herein is a host cell comprising the nucleicacid described herein. In another aspect, provided herein is a methodfor producing an antibody described herein comprising culturing the hostcell described herein under conditions suitable for production of theantibody. In certain embodiments, the method for producing an antibodydescribed herein further comprises recovering the antibody describedherein produced by the host cell described herein. In another aspect,provided herein is an antibody produced by the method for producing anantibody described herein. In another aspect, provided herein is apharmaceutical composition comprising any of the antibodies describedherein and a pharmaceutically acceptable carrier.

In yet another aspect, provided herein is a method for treating orpreventing a disease or disorder mediated by granulocytes in anindividual comprising administering to the individual an effectiveamount of any of the antibodies described herein. In certainembodiments, the antibody specifically binds an NSP4 active site and/orinhibits catalytic activity of NSP4. In certain embodiments, theantibody specifically binds an NSP4 heparin binding site and/or competeswith heparin for binding to NSP4. In certain embodiments, an effectiveamount of an antibody that specifically binds an NSP4 active site and/orinhibits catalytic activity of NSP4 and an antibody that specificallybinds an NSP4 heparin binding site and/or competes with heparin forbinding to NSP4 is administered to the individual. In certainembodiments that may be combined with any of the preceding embodiments,the disease or disorder is a neutrophil-mediated, aneosinophil-mediated, or a basophil-mediated disease or disorder. Incertain embodiments, the neutrophil-mediated disease or disorder isselected from vascular disease and inflammatory disease. In certainembodiments, the vascular disease is selected from stroke, diabeticretinopathy, edema, diabetic macular edema, hereditary angioedema,idiopathic angioedema, leakage of vasculature, and cerebral ischemia. Incertain embodiments, the inflammatory disease is selected from acutelung injury, asthma, chronic obstructive pulmonary disease (COPD), acuterespiratory distress syndrome (ARDS), osteoarthritis, rheumatoidarthritis, and septic shock. In certain embodiments, the disease ordisorder is selected from stroke, diabetic retinopathy, edema, diabeticmacular edema, hereditary angioedema, idiopathic angioedema, leakage ofvasculature, systemic lupus erythematosus (SLE), autoimmunevasculitides, cerebral ischemia, acute lung injury, anaphylaxis,systemic anaphylaxis, allergic lung inflammation, idiopathic lungfibrosis, asthma, allergic asthma, virus-induced asthma, chronicobstructive pulmonary disease (COPD), acute respiratory distresssyndrome (ARDS), osteoarthritis, rheumatoid arthritis, psoriasis,psoriatic arthritis, blistering skin disease, bullous pemphigoid,inflammatory skin disease, atopic dermatitis, urticaria, eosinophiliccellulitis, cancer, lung cancer, kidney disease, glomerulonephritis,septic shock, inflammatory bowel disease, ulcerative colitis, andCrohn's disease. In certain embodiments that may be combined with any ofthe preceding embodiments, the individual is a human. In certainembodiments that may be combined with any of the preceding embodiments,the antibody is administered intravenously, intramuscularly,subcutaneously, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. In certain embodiments that may becombined with any of the preceding embodiments, the anti-NSP4 antibodyis formulated in a pharmaceutical composition comprising the antibodyand a pharmaceutically acceptable carrier.

In yet another aspect, provided herein is an article of manufacturecomprising any of the antibodies described herein. In certainembodiments, the article of manufacture further comprises a packageinsert comprising instructions for using the antibody to treat orprevent a disease or disorder mediated by granulocytes in an individual.In some embodiments, the disease or disorder is an eosinophil-mediated,basophil-mediated, or a neutrophil-mediated disease or disorder. In someembodiments, the disease or disorder that can be treated by an antibodydescribed herein is a vascular disease, an inflammatory disease, or anautoimmune disease. In some embodiments, the disease or disorder isselected from the group consisting of stroke, diabetic retinopathy,edema, diabetic macular edema, hereditary angioedema, idiopathicangioedema, leakage of vasculature, cerebral ischemia, acute lunginjury, anaphylaxis, systemic anaphylaxis, allergic lung inflammation,asthma (e.g., allergic asthma, virus-induced asthma), chronicobstructive pulmonary disease (COPD), acute respiratory distresssyndrome (ARDS), idiopathic pulmonary fibrosis, systemic lupuserythematosus (SLE), autoimmune vasculitides, blistering skin diseases(e.g., bullous pemphigoid), inflammatory skin diseases (e.g., atopicdermatitis, urticarial, eosinophilic cellulitis), cancer (e.g., lungcancer), kidney diseases (e.g., glomerulonephritis), osteoarthritis,rheumatoid arthritis, psoriatic arthritis, psoriasis, septic shock,inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease).In some embodiments, the individual has the disease or disorder or hasbeen diagnosed with the disease or disorder. In some embodiments, theindividual is at risk of developing the disease or disorder. In someembodiments, the individual is a human.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

DESCRIPTION OF THE FIGURES

FIG. 1 is a sequence alignment of the active region of NSP4 orthologs.Residues are numbered at the top using the chymotrypsinogen numberingsystem. The disulfide bonds are denoted at the bottom with lines.Conserved residues are colored using the ClustalX scheme. NSP4 primarysubstrate specificity determinants at the 190, 192, and 216 positionsare labeled and outlined. The length of the 180-loop (¹⁹⁰FCS¹⁹²) and220-loop (²¹⁴SFSGxxC²²⁰) segments that coordinate the P1-arginine sidechain is also conserved in NSP4 orthologs and is denoted with black barsat the top. Two or more NSP4 paralogs are found in the Tasmanian devil,frogs (X. laevis and X. tropicalis), and coelacanth; some paralogs havesubstitutions at the 192 position, but all species have at least oneparalog that have all the requisite H-bond acceptors. Reptilian andavian species have heterophils instead of bona fide neutrophils, andtheir NSP4 orthologs have a F190V substitution. Avian NSP4 orthologs arepredicted to have reduced activity due to a S192A substitution and havea lone unpaired C136 because of a C201F substitution. NSP4 orthologsequences were compiled from National Center of BiotechnologyInformation (NCBI) and ENSEMBL databases and aligned using ClustalW(Larkin et al., Bioinformatics, 2007, 23:2947-2948). The preliminaryalignment was then manually curated and analyzed using SeaView (Gouy etal., Mol Biol Evol, 2010, 27:221-224) and Unipro UGENE (Okonechnikov etal., Bioinformatics, 2012, 28:1166-1167).

FIGS. 2A-2D demonstrate that NSP4 has an elastase-like active site withtrypsin-like arginine specificity. A) Schematic of the NSP4 S1 pocket.B) Schematic of trypsin, chymotrypsin, and neutrophil elastase S1pockets and their interactions with the preferred P1 residue.Trypsin-like proteases have a deep S1 pocket and form a salt bridgeinteraction between the substrate P1-arginine and the conserved D189 atthe base of the S1 pocket (dashed lines). Chymotrypsin-like proteaseshave a large hydrophobic pocket to accommodate bulky P1 residues.Elastase-like proteases have a shallow S1 pocket, formed by residues atthe 216 and 190 positions, to accommodate small aliphatic P1 residues.C) Profiling of NSP4 P1 substrate specificity by use of a fluorogenicpeptide substrate panel with different P1 residues treated withrecombinant wildtype (WT) or the inactive (S195A) NSP4 produced frominsect (Baculovirus Expression vector systems; BEVS) or mammalian cells(Chinese Hamster Ovarian; CHO). P1-Arg peptide: WT (BEVS)k_(cat)/K_(M)=1.1×10⁴±2.1×10³ M⁻¹ s⁻¹ and WT (CHO)k_(cat)/K_(M)=7.5×10³±1.1×10³ M⁻¹s⁻¹. D) Cleavage of fluorogenic peptideby NSP4 S1 pocket mutants. Activity is measured as nanomolar productformed per second (nM/s). Results are mean±standard deviation from atleast three independent experiments.

FIGS. 3A-3B demonstrate that NSP4 features an occluded S1 pocket andnon-canonical interactions with P1-arginine. A) Superposition of NSP4crystal structures: NSP4-apo form 1, NSP4-apo form 2, NSP4:FFR-cmk, andNSP4:VLK-cmk. Asterisks denote catalytic triad residues. B) Top left,FFR-cmk (sticks) bound to the canonical trypsin-like protease, factorVIIa, PDB 1DAN. Top right, FFR-cmk (sticks) bound to NSP4. Bottom left,side view of canonical S1 pockets with superposition of ten differentP1-arginine ligands (sticks) bound to trypsin-like proteases (ribbons).Structures shown are 1DAN, 1SHH, 1ORF, 1AUT, 1PFX, 2FIR, 1LMW, 1BUI,2B80, 1BDA. Bottom right, side view of FFR-cmk (sticks) bound to NSP4.

FIGS. 4A-4B demonstrate that NSP4 possesses a basic patch that bindsheparin via ionic interactions. A) Surface representation of NSP4 asshaded by Poisson-Boltzmann electrostatic calculations contoured from −5to 5 kT/e. S1 denotes the occluded S1 pocket. B) NSP4 binding tofluorescein-conjugated heparin determined by fluorescence polarizationat different ionic conditions (left) and by native polyacrylamide gelelectrophoresis at the 0.15M NaCl condition (right).

FIG. 5 shows the representative electron density at the NSP4 activesite. σA-weighted 2mFo-DFc electron density map, contoured at 1.5σ, ofthe NSP4 active site. Shown are the 220-loop residues ²¹⁴SFSGLWC²²⁰, the180-loop residues ¹⁹⁰ FCS¹⁹², and the catalytic residues H57 and S195.The S216 and F190 residues, which occlude the S1 pocket, are shown assticks. The bound FFR-cmk and VLK-cmk are shown as sticks.

FIGS. 6A-6D demonstrate that the P1-arginine in the “up” confirmation isstabilized by a H-bond network. A) Structural superposition ofP1-arginine in NSP4 and in Factor VIIa (PDB 1DAN). B) H-bond networkaround P1-arginine residue (P1-Arg) in NSP4. C) Cleavage of fluorogenicpeptide by NSP4 hydrogen bond mutants. D) Cleavage of fluorogenicpeptides with P1-arginine and P1-methylarginine by NSP4 or Factor Xa(FXa).

FIG. 7 demonstrates the structural basis for NSP4 preference forP1-arginine over P1-lysine. The P1-arginine in NSP4:FFR-cmk structure isstabilized by a network of H-bonds, coordinated by the H-bond acceptorsS216 and S192 side chains and G217 backbone carbonyl oxygen. Incontrast, the P1-lysine in NSP4:VLK-cmk structure does not engage thefull complement of H-bond acceptors on NSP4.

FIG. 8 shows confirmation of NSP4 or FXa cleavage of modifiedP1-arginine fluorogenic peptide substrates by liquid chromatography-massspectrometry (LC-MS). Cleavage of P1-arginine (P1-Arg, left panel) orP1-methylarginine (P1-Arg(Me), right panel) fluorogenic peptidesubstrate by NSP4 (50 nM) or by factor Xa (FXa, 200 nM) was confirmed byuse of LC-MS. The peaks, measured in total ion current, representseither full-length or hydrolyzed peptide fragments separated overreverse-phase HPLC. The identities of peptide fragments and theirdeduced mass are indicated. P1-Arg(Me) is refractory to FXa cleavage;the peaks observed in P1-Arg(Me) peptides for FXa represent a shifted P1cleavage event occurring at ¹IR²↓³{Arg(Me)}SSYSFKK¹⁰ instead of the¹IR{Arg(Me)}³↓⁴SSYSFKK¹⁰ observed for NSP4.

FIGS. 9A-9C show the characterization of NSP4^(−/−) mice. A) Targetingstrategy for generating NSP4^(−/−) mice by homologous recombination, aspreviously described (Tang et al., Nat Biotechnol, 2010, 28:749-755). B)NSP4 ablation in NSP4^(−/−) mice was confirmed by RT-qPCR from totalbone marrow cells using three distinct NSP4 primers (PRSS57_1, PRSS57_2,PRSS57_3). The expression of neighboring protease genes, neutrophilelastase (Elane), granzyme M (Gzmm), proteinase 3 (Prtn3), andcomplement factor D (Cfd) were also analyzed. All gene expression levelsare quantified by its relative abundance to mammalian 18S rRNA (dCt). C)Counting of viable total bone marrow cells and sorted B cells (B220⁺),myeloid cells (B220⁻/CD11b⁺), monocytes (B220⁻/CD11b⁺/Ly6C^(hi)) andneutrophils (B220⁻/CD11b⁺/Ly6G^(hi)) cells.

FIGS. 10A-10B show that NSP4 is required for K/B×N serum-inducedvascular leakage. The fluorescence probe AngioSense 680 was injectedintravenously via tail vein (at −5 min), allowed to equilibrate for 5minutes, followed by intravenous injection of the K/B×N arthritic serum(at 0 min). A) Representative near-infrared fluorescence imaging of theforepaws from a wild-type and a NSP4^(−/−) mouse. Vascular leakage couldbe observed in the wild-type mouse forepaw starting at 5 min (arrows).B) Individual quantification of the fold change in mean fluorescenceintensity of wild-type mice or NSP4^(−/−) mice, as labeled, n=5 mice pergroup.

FIGS. 11A-11C show that NSP4 is required for K/B×N serum-inducedarthritis. A) Clinical scores of mouse paws following systemic K/B×Nserum injection in wild-type (circles) and NSP4^(−/−) (squares) mice.The data is represented as mean±SEM, n=4 mice per group. B) Histologicallesion scores of mouse paws at the experimental end point. All four pawsof each mouse, n=4 mice per group, were examined as hemisections andscored for inflammation, fibroplasia, cartilage injury and boneremodeling on a scale of 0 (no lesions) to 5 (severe lesions). Wild-typemice had mild to moderate lesions (mean histological lesion scores of3.4, 3.0, 2.1, 2.2), whereas NSP4^(−/−) mice were essentially protected(mean histological lesion scores of 0.70, 0.80, 0.00, 0.78). Group-wisecomparison on all four histological lesion score categories reachedstatistical significance with p<0.0001. C) Representative histologicalsections of wild-type (left) and NSP4^(−/−) (right) mouse forepawsstained with hematoxylin and eosin. Top: wild-type mice had-considerablesoft tissue swelling with extensive periarticular inflammatory cellinfiltration (arrows) and fibroplasia. Middle: wild-type paw lesionsincluded intraarticular exudate (asterisk), periosteal osteolysis(arrowheads), and inflammatory cell infiltration (double arrows).Bottom: wild-type mice had dense periarticular inflammatory cellinfiltration. NSP4^(−/−) mice exhibited no tissue swelling (top),maintained normal joint architecture (middle), and had minimal cellularinfiltrate (bottom).

FIG. 12 is a diagram of a precursor form of NSP4 (zymogen) and matureform of NSP4 (active) used for generation of conformation-specific NSP4antibodies by phage display. hZ indicates precursor form of human NSP4;mZ indicates precursor form of mouse NSP4; hA indicates mature form ofhuman NSP4; and mA indicates mature form of mouse NSP4.

FIG. 13 is a diagram of the CDR sequences of the heavy chain variableregion of antibodies 5-2, 5-3, and 5-4. The sequences depicted in thefigure are identified in the sequence listing as follows: CDRH1 forphage clone 5-2, SEQ ID NO:1; CDRH2 for phage clone 5-2, SEQ ID NO:2;CDRH3 for phage clone 5-2, SEQ ID NO:3; CDRH1 for phage clone 5-3, SEQID NO:4; CDRH2 for phage clone 5-3, SEQ ID NO:5; CDRH3 for phage clone5-3, SEQ ID NO:6; CDRH1 for phage clone 5-4, SEQ ID NO:7; CDRH2 forphage clone 5-4, SEQ ID NO:8; and CDRH3 for phage clone 5-4, SEQ IDNO:9.

FIGS. 14A-14C show a series of graphs showing specific binding ofantibodies A) 5-2, B) 5-3, and C) 5-4 to the mature form of mouse NSP4in biolayer interferometry experiments. hZ indicates precursor form ofhuman NSP4; mZ indicates precursor form of mouse NSP4; hA indicatesmature form of human NSP4; and mA indicates mature form of mouse NSP4.

FIG. 15 is a graph demonstrating antibodies 5-2, 5-3, and 5-4specifically blocked the protease activity of mouse NSP4 in afluorogenic substrate assay. hNSP indicates mature human NSP4. mNSP4indicates mature mouse NSP4.

FIGS. 16A-16C show a series of graphs showing the mRNA measurement offour distinct sorted bone marrow cell fractions isolated from fourwild-type (WT) and four NSP4-deficient mice (NSP4 PRSS57 KO). Thetranscript levels in wild-type mice are denoted by circles and thetranscript levels in NSP4-deficient mice are denoted by squares. Threedifferent NSP4/Prss57 primer/probe sets that span three different exonjunctions were used: A) spans exons 1-2, B) spans exons 2-3, and C)spans exons 3-4.

FIG. 17 is a series of Western blots performed to detect the presence ofNSP4 protein in four different mouse myeloid lineage cell types(neutrophils, eosinophils, mast cells, and macrophages). Equal amountsof protein lysates (10 ug/lane) were loaded and NSP4 was detected usinga rabbit anti-mouse NSP4 polyclonal antibody. Mouse bone marrow-derivedneutrophil, eosinophil, mast cells, and macrophages were isolated fromthree wild-type (WT) and three NSP4 (Prss57)-deficient (KO) mice bonemarrows.

FIGS. 18A-18C show that NSP4 is necessary for neutrophil recruitment. A)Luminol-based bioluminescence imaging of myeloperoxidase (MPO) activityof mouse paws following systemic K/B×N serum injection at the indicatedtime points. Wildtype (WT) or NSP4-deficient (KO) mice were tested, asindicated, and quantified as photon counts per minute (cpm) per cm². B)Western blot of MPO and control protein beta actin using total mousebone marrow-derived neutrophil lysates isolated from wild-type (WT) andNSP4-deficient (KO) mice. Each lane represented pooled lysates fromthree mice per genotype. C) Representative graph of luminol-basedbioluminescence of mouse paws following K/B×N serum injection inwildtype (WT) or NSP4-deficient (NSP4KO) mice (n=3).

FIG. 19 shows the estimation of NSP4 antibody clone affinity by phageIC50 assay. Detection of phage clones preincubated with soluble NSP4protein before placement into wells coated with 2 ug/ml of NSP4 andsubsequent washing of wells with phosphate buffered saline supplementedwith 0.02% Tween-20. hZ, human zymogen NSP4; mZ, mouse zymogen NSP4; hA,human active NSP4; mA, mouse active NSP4. Higher affinity clones bind tosoluble NSP4 at lower soluble NSP4 concentrations and thus have curvesshifted to the left.

FIGS. 20A-20B show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 1-1 (A) and 1-2 (B) (both in human IgG1 format)binding to different variants of purified NSP4 protein. hZ, humanzymogen NSP4; mZ, mouse zymogen NSP4; hA, human active NSP4; mA, mouseactive NSP4; all as labeled.

FIGS. 21A-21B show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 1-3 (A) and 1-5 (B) (both in human IgG1 format)binding to different variants of purified NSP4 protein. hZ, humanzymogen NSP4; mZ, mouse zymogen NSP4; hA, human active NSP4; mA, mouseactive NSP4; all as labeled.

FIGS. 22A-22D show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 2-1 (A), 2-2 (B), 2-3 (C), and 2-4 (D) (all inhuman IgG1 format) binding to different variants of purified NSP4protein. hZ, human zymogen NSP4; mZ, mouse zymogen NSP4; hA, humanactive NSP4; mA, mouse active NSP4; all as labeled.

FIG. 23 shows the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibody 2-5 (in human IgG1 format) binding to differentvariants of purified NSP4 protein. hZ, human zymogen NSP4; mZ, mousezymogen NSP4; hA, human active NSP4; mA, mouse active NSP4; all aslabeled.

FIGS. 24A-24B show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 3-2 (A) and 3-5 (B) (both in human IgG1 format)binding to different variants of purified NSP4 protein. hZ, humanzymogen NSP4; mZ, mouse zymogen NSP4; hA, human active NSP4; mA, mouseactive NSP4; all as labeled.

FIGS. 25A-25C show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 4-2 (A), 4-3 (B), and 4-4 (C) (all in humanIgG1 format) binding to different variants of purified NSP4 protein. hZ,human zymogen NSP4; mZ, mouse zymogen NSP4; hA, human active NSP4; mA,mouse active NSP4; all as labeled.

FIGS. 26A-26D show the determination of NSP4 antibody clone affinity bybiolayer interferometry. Biolayer interferometry measurement of purifiedNSP4-specific antibodies 5-1 (A), 5-2 (B), 5-3 (C), and 5-4 (D) (all inhuman IgG1 format) binding to different variants of purified NSP4protein. hZ, human zymogen NSP4; mZ, mouse zymogen NSP4; hA, humanactive NSP4; mA, mouse active NSP4; all as labeled.

FIG. 27 shows the screening of antibodies panned against zymogen NSP4 byfluorogenic activity assay using active NSP4. Fluorogenic peptidecleavage assay of purified human active NSP4 (gray bars), mouse activeNSP4 (white bars), or buffer alone (no enzyme; black bars) preincubatedwith either buffer alone (“No Ab”) or different purified anti-NSP4antibodies.

FIG. 28 shows the screening for NSP4 blocking antibodies by fluorogenicactivity assay using active NSP4. Fluorogenic peptide cleavage assay ofpurified human active NSP4 (gray bars), mouse active NSP4 (white bars),or buffer alone (no enzyme; black bars) preincubated with either bufferalone (“No Ab”) or different purified NSP4-specific antibodies.Asterisks denote antibodies that inhibit NSP4 enzyme activity.

FIG. 29 is a summary of NSP4 antibody characterization. Provided is asummary of in vitro characterization of purified NSP4-specific antibodyas determined by (from left to right): affinity to purified NSP4variants by biolayer interferometry, specificity classification asdetermined by biolayer interferometry, and blocking antibody screen byfluorogenic enzyme activity assay.

FIG. 30 shows epitope mapping of NSP4 antibodies with Fab 5-1. Epitopemapping of mouse-specific NSP4 antibodies by biolayer interferometrymeasurements of purified NSP4-specific antibodies binding to purifiedmouse NSP4 in the presence of Fab 5-1.

FIG. 31 shows epitope mapping of NSP4 antibodies with heparin. Epitopemapping of mouse-specific NSP4 antibodies by biolayer interferometrymeasurements of purified NSP4-specific antibodies binding to purifiedmouse NSP4 in the presence of heparin sulfate.

FIG. 32 shows the affinity maturation of the pan NSP4 Ab35 antibodyclone. Affinity improvements of antibodies based on Ab 3-5 (“35.WT”) asdetermined by phage IC50 assay using soluble mNSP4 (top) or solublehNSP4 (bottom) competitor.

FIG. 33 shows the affinity maturation of the conformation-specific NSP4Ab51 antibody clone. Affinity improvements of antibodies based on Ab 5-1(“51.WT”) as determined by phage IC50 assay using soluble mNSP4competitor.

DETAILED DESCRIPTION I. Definitions

The terms “neutrophil serine protease 4” of “NSP4” as used herein, referto any native NSP4 from any mammals such as primates (e.g., human,rhesus, chimpanzee NSP4) and rodents (e.g., mice and rats), unlessotherwise indicated. The term encompasses “full-length,” unprocessedNSP4 such as a precursor or zymogen form of NSP4 as well as any form ofNSP4 that results from proteolytic cleavage such as a mature or activeform of NSP4. The term also encompasses naturally occurring variants ofNSP4, e.g., splice variants or allelic variants. The amino acid sequenceof an exemplary human NSP4 is shown in SEQ ID NO:17. The amino acidsequence of another exemplary human NSP4 is shown in SEQ ID NO:18.

Human NSP4 sequence (SEQ ID NO: 17)MGLGLRGWGRPLLTVATALMLPVKPPAGSWGAQIIGGHEVTPHSRPYMASVRFGGQHHCGGFLLRARWVVSAAHCFSHRDLRTGLVVLGAHVLSTAEPTQQVFGIDALTTHPDYHPMTHANDICLLRLNGSAVLGPAVGLLRLPGRRARPPTAGTRCRVAGWGFVSDFEELPPGLMEAKVRVLDPDVCNSSWKGHLTLTMLCTRSGDSHRRGFCSADSGGPLVCRNRAHGLVSFSGLWCGDPKTPDVYTQVSAFVAWIWDVVRRSSPQPGPLPGTTRPPGEAA Human NSP4 sequence (SEQ ID NO: 18)MGLGLRGWGRPLLTVATALMLPVKPPAGSWGAQIIGGHEVTPHSRPYMASVRFGGQHHCGGFLLRARWVVSAAHCFSHRDLRTGLVVLGAHVLSTAEPTQQVFGIDALTTHPDYHPMTHANDICLLRLNGSAVLGPAVGLLRPPGRRARPPTAGTRCRVAGWGFVSDFEELPPGLMEAKVRVLDPDVCNSSWKGHLTLTMLCTRSGDSHRRGFCSADSGGPLVCRNRAHGLVSFSGLWCGDPKTPDVYTQVSAFVAWIWDVVRRSSPQPGPLPGTTRPPGEAA

The term “neutrophil serine protease 4 inhibitor” or “NSP4 inhibitor,”as used herein, refers to a molecule that blocks, inhibits, reduces(including significantly), or interferes with a NSP4 (mammalian, such asa human NSP4) biological activity in vitro, in situ, and/or in vivo. Theterm “inhibitor” implies no specific mechanism of biological actionwhatsoever, and expressly includes and encompasses all possiblepharmacological, physiological, and biochemical interactions with a NSP4whether direct or indirect, and whether interacting with a NSP4, itssubstrate, or through another mechanism, and its consequences which canbe achieved by a variety of different, and chemically divergent,compositions. Exemplary NSP4 inhibitors include, but are not limited to,an anti-NSP4 antibody that specifically binds to a NSP4 or one or boththe precursor form and mature form of a NSP4, an anti-sense moleculedirected to a nucleic acid encoding a NSP4, a short interfering RNA(“siRNA”) molecule directed to a nucleic acid encoding a NSP4, a NSP4inhibitory compound, an RNA or DNA aptamer that binds to a NSP4 or oneor both the precursor form and mature form of a NSP4, and a NSP4structural analog. In some embodiments, a NSP4 inhibitor (e.g., anantibody) binds (physically interacts with) a NSP4, binds to a NSP4substrate, and/or inhibits (reduces) NSP4 synthesis, production orrelease. In other embodiments, a NSP4 inhibitor binds a NSP4 andprevents its binding to its substrate. In still other embodiments, aNSP4 inhibitor reduces or eliminates expression (i.e., transcription ortranslation) or proteolytic processing of a NSP4. Examples of types ofNSP4 inhibitors are provided herein.

As used herein, the term “RNA interference” or “RNAi” refers generallyto a process in which a double-stranded RNA molecule or a short hairpinRNA molecule reducing or inhibiting the expression of a nucleic acidsequence with which the double-stranded or short hairpin RNA moleculeshares substantial or total homology. The term “short interfering RNA”or “siRNA” or “RNAi agent” refers to an RNA sequence that elicits RNAinterference. See Kreutzer et al., WO 00/44895; Zernicka-Goetz et al.,WO 01/36646; Fire, WO 99/32619; Mello and Fire, WO 01/29058. As usedherein, siRNA molecules include RNA molecules encompassing chemicallymodified nucleotides and non-nucleotides. The term “ddRNAi agent” refersto a DNA-directed RNAi agent that is transcribed from an exogenousvector. The terms “short hairpin RNA” or “shRNA” refer to an RNAstructure having a duplex region and a loop region. In certainembodiments, ddRNAi agents are expressed initially as shRNAs.

As used herein, the term “aptamer” refers to a heterologousoligonucleotide capable of binding tightly and specifically to a desiredmolecular target, such as, for example, common metabolic cofactors(e.g., Coenzyme A, S-adenosyl methionine, and the like), proteins (e.g.,complement protein C5, antibodies, and the like), or conservedstructural elements in nucleic acid molecules (e.g., structuresimportant for binding of transcription factors and the like). Aptamerstypically comprise DNA or RNA nucleotide sequences ranging from about 10to about 100 nucleotides in length, from about 10 to about 75nucleotides in length, from about 10 to about 50 nucleotides in length,from about 10 to about 35 nucleotides in length, and from about 10 toabout 25 nucleotides in length. Synthetic DNA or RNA oligonucleotidescan be made using standard solid phase phosphoramidite methods andequipment, such as by using a 3900 High Throughput DNA Synthesizer™available from Applied Biosystems (Foster City, Calif.). Aptamersfrequently incorporate derivatives or analogs of the commonly occurringnucleotides found in DNA and RNA (e.g., A, G, C, and T/U), includingbackbone or linkage modifications (e.g., peptide nucleic acid (PNA) orphosphothioate linkages) to increase resistance to nucleases, bindingavidity, or to otherwise alter their pharmacokinetic properties.Exemplary modifications are set forth in U.S. Pat. Nos. 6,455,308;4,469,863; 5,536,821; 5,541,306; 5,637,683; 5,637,684; 5,700,922;5,717,083; 5,719,262; 5,739,308; 5,773,601; 5,886,165; 5,929,226;5,977,296; 6,140,482; and in WIPO publications WO 00/56746 and WO01/14398. Methods for synthesizing oligonucleotides comprising suchanalogs or derivatives are disclosed, for example, in the patentpublications cited above, and in U.S. Pat. Nos. 6,455,308; 5,614,622;5,739,314; 5,955,599; 5,962,674; 6,117,992; and in WO 00/75372.

A “blocking” antibody is one that inhibits or reduces a biologicalactivity of the antigen it binds. In some embodiments, blockingantibodies substantially or completely inhibit the biological activityof the antigen. In some embodiments, the antigen is a NSP4.

The terms “anti-NSP4 antibody” and “an antibody that binds to NSP4”refer to an antibody that is capable of binding a NSP4 with sufficientaffinity such that the antibody is useful as a diagnostic and/ortherapeutic agent in targeting a NSP4. In one embodiment, the extent ofbinding of an anti-NSP4 antibody to an unrelated, non-NSP4 protein isless than about 10% of the binding of the antibody to NSP4 as measured,e.g., by a radioimmunoassay (RIA) or enzyme-linked immunosorbent assay(ELISA). In certain embodiments, an antibody that binds to a NSP4 has adissociation constant (Kd) of ≤104, ≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM,≤0.01 nM, or ≤0.001 nM (e.g., 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M,e.g., from 10⁻⁹M to 10⁻¹³ M). In certain embodiments, an anti-NSP4antibody binds to an epitope of NSP4 that is conserved among NSP4 fromdifferent species.

The term “antibody” herein is used in the broadest sense and encompassesvarious antibody structures, including but not limited to monoclonalantibodies, polyclonal antibodies, multispecific antibodies (e.g.,bispecific antibodies), and antibody fragments so long as they exhibitthe desired antigen-binding activity.

An “antibody fragment” refers to a molecule other than an intactantibody that comprises a portion of an intact antibody that binds theantigen to which the intact antibody binds. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)2, diabodies, linear antibodies, single-chain antibody molecules(e.g., scFv), and multispecific antibodies formed from antibodyfragments.

An “antibody that binds to the same epitope” as a reference antibodyrefers to an antibody that blocks binding of the reference antibody toits antigen in a competition assay by 50% or more, and conversely, thereference antibody blocks binding of the antibody to its antigen in acompetition assay by 50% or more. An exemplary competition assay isprovided herein.

The term “chimeric” antibody refers to an antibody in which a portion ofthe heavy and/or light chain is derived from a particular source orspecies, while the remainder of the heavy and/or light chain is derivedfrom a different source or species.

The “class” of an antibody refers to the type of constant domain orconstant region possessed by its heavy chain. There are five majorclasses of antibodies: IgA, IgD, IgE, IgG, and IgM, and several of thesemay be further divided into subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains thatcorrespond to the different classes of immunoglobulins are called α, δ,ε, γ, and μ, respectively.

“Effector functions” refer to those biological activities attributableto the Fc region of an antibody, which vary with the antibody isotype.Examples of antibody effector functions include: C1q binding andcomplement dependent cytotoxicity (CDC); Fc receptor binding;antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g. B cell receptor); and B cellactivation.

An “acceptor human framework” for the purposes herein is a frameworkcomprising the amino acid sequence of a light chain variable domain (VL)framework or a heavy chain variable domain (VH) framework derived from ahuman immunoglobulin framework or a human consensus framework, asdefined below. An acceptor human framework “derived from” a humanimmunoglobulin framework or a human consensus framework may comprise thesame amino acid sequence thereof, or it may contain amino acid sequencechanges. In some embodiments, the number of amino acid changes are 10 orless, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less,3 or less, or 2 or less. In some embodiments, the VL acceptor humanframework is identical in sequence to the VL human immunoglobulinframework sequence or human consensus framework sequence.

“Affinity” refers to the strength of the sum total of noncovalentinteractions between a single binding site of a molecule (e.g., anantibody) and its binding partner (e.g., an antigen). Unless indicatedotherwise, as used herein, “binding affinity” refers to intrinsicbinding affinity which reflects a 1:1 interaction between members of abinding pair (e.g., antibody and antigen). The affinity of a molecule Xfor its partner Y can generally be represented by the dissociationconstant (Kd). Affinity can be measured by common methods known in theart, including those described herein. Specific illustrative andexemplary embodiments for measuring binding affinity are described inthe following.

An “affinity matured” antibody refers to an antibody with one or morealterations in one or more hypervariable regions (HVRs), compared to aparent antibody which does not possess such alterations, suchalterations resulting in an improvement in the affinity of the antibodyfor antigen.

The term “Fc region” herein is used to define a C-terminal region of animmunoglobulin heavy chain that contains at least a portion of theconstant region. The term includes native sequence Fc regions andvariant Fc regions. In one embodiment, a human IgG heavy chain Fc regionextends from Cys226, or from Pro230, to the carboxyl-terminus of theheavy chain. However, the C-terminal lysine (Lys447) of the Fc regionmay or may not be present. Unless otherwise specified herein, numberingof amino acid residues in the Fc region or constant region is accordingto the EU numbering system, also called the EU index, as described inKabat et al., Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.,1991.

“Framework” or “FR” refers to variable domain residues other thanhypervariable region (HVR) residues. The FR of a variable domaingenerally consists of four FR domains: FR1, FR2, FR3, and FR4.Accordingly, the HVR and FR sequences generally appear in the followingsequence in VH (or VL): FR1-H1(L1)-FR2-H2(L2)-FR3-H3(L3)-FR4.

The terms “full length antibody,” “intact antibody,” and “wholeantibody” are used herein interchangeably to refer to an antibody havinga structure substantially similar to a native antibody structure orhaving heavy chains that contain an Fc region as defined herein.

The terms “host cell,” “host cell line,” and “host cell culture” areused interchangeably and refer to cells into which exogenous nucleicacid has been introduced, including the progeny of such cells. Hostcells include “transformants” and “transformed cells,” which include theprimary transformed cell and progeny derived therefrom without regard tothe number of passages. Progeny may not be completely identical innucleic acid content to a parent cell, but may contain mutations. Mutantprogeny that have the same function or biological activity as screenedor selected for in the originally transformed cell are included herein.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human or a human cellor derived from a non-human source that utilizes human antibodyrepertoires or other human antibody-encoding sequences. This definitionof a human antibody specifically excludes a humanized antibodycomprising non-human antigen-binding residues.

A “human consensus framework” is a framework which represents the mostcommonly occurring amino acid residues in a selection of humanimmunoglobulin VL or VH framework sequences. Generally, the selection ofhuman immunoglobulin VL or VH sequences is from a subgroup of variabledomain sequences. Generally, the subgroup of sequences is a subgroup asin Kabat et al., Sequences of Proteins of Immunological Interest, FifthEdition, NIH Publication 91-3242, Bethesda Md. (1991), vols. 1-3. In oneembodiment, for the VL, the subgroup is subgroup kappa I as in Kabat etal., supra. In one embodiment, for the VH, the subgroup is subgroup IIIas in Kabat et al., supra.

A “humanized” antibody refers to a chimeric antibody comprising aminoacid residues from non-human HVRs and amino acid residues from humanFRs. In certain embodiments, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the HVRs (e.g., CDRs) correspond tothose of a non-human antibody, and all or substantially all of the FRscorrespond to those of a human antibody. A humanized antibody optionallymay comprise at least a portion of an antibody constant region derivedfrom a human antibody. A “humanized form” of an antibody, e.g., anon-human antibody, refers to an antibody that has undergonehumanization.

The term “hypervariable region” or “HVR,” as used herein, refers to eachof the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops (“hypervariable loops”).Generally, native four-chain antibodies comprise six HVRs; three in theVH (H1, H2, H3), and three in the VL (L1, L2, L3). HVRs generallycomprise amino acid residues from the hypervariable loops and/or fromthe “complementarity determining regions” (CDRs), the latter being ofhighest sequence variability and/or involved in antigen recognition. AnHVR region as used herein comprise any number of residues located withinpositions 24-36 (for L1), 46-56 (for L2), 89-97 (for L3), 26-35B (forH1), 47-65 (for H2), and 93-102 (for H3). Therefore, an HVR includesresidues in positions described previously:

A) 24-34 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and96-101 (H3) (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987);

B) 24-34 of L1, 50-56 of L2, 89-97 of L3, 31-35B of H1, 50-65 of H2, and95-102 of H3 (Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991).

C) 30-36 (L1), 46-55 (L2), 89-96 (L3), 30-35 (H1), 47-58 (H2), 93-100a-j(H3) (MacCallum et al. J. Mol. Biol. 262:732-745 (1996).

With the exception of CDR1 in VH, CDRs generally comprise the amino acidresidues that form the hypervariable loops. CDRs also comprise“specificity determining residues,” or “SDRs,” which are residues thatcontact antigen. SDRs are contained within regions of the CDRs calledabbreviated-CDRs, or a-CDRs. Exemplary a-CDRs (a-CDR-L1, a-CDR-L2,a-CDR-L3, a-CDR-H1, a-CDR-H2, and a-CDR-H3) occur at amino acid residues31-34 of L1, 50-55 of L2, 89-96 of L3, 31-35B of H1, 50-58 of H2, and95-102 of H3. (See Almagro and Fransson, Front. Biosci. 13:1619-1633(2008).) Unless otherwise indicated, HVR residues and other residues inthe variable domain (e.g., FR residues) are numbered herein according toKabat et al., supra.

The term “variable region” or “variable domain” refers to the domain ofan antibody heavy or light chain that is involved in binding theantibody to antigen. The variable domains of the heavy chain and lightchain (VH and VL, respectively) of a native antibody generally havesimilar structures, with each domain comprising four conserved frameworkregions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindtet al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).)A single VH or VL domain may be sufficient to confer antigen-bindingspecificity. Furthermore, antibodies that bind a particular antigen maybe isolated using a VH or VL domain from an antibody that binds theantigen to screen a library of complementary VL or VH domains,respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887(1993); Clarkson et al., Nature 352:624-628 (1991).

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

An “isolated” antibody is one which has been separated from a componentof its natural environment. In some embodiments, an antibody is purifiedto greater than 95% or 99% purity as determined by, for example,electrophoretic (e.g., SDS-PAGE, isoelectric focusing (IEF), capillaryelectrophoresis) or chromatographic (e.g., ion exchange or reverse phaseHPLC). For review of methods for assessment of antibody purity, see,e.g., Flatman et al., J. Chromatogr. B 848:79-87 (2007).

An “isolated” nucleic acid refers to a nucleic acid molecule that hasbeen separated from a component of its natural environment. An isolatednucleic acid includes a nucleic acid molecule contained in cells thatordinarily contain the nucleic acid molecule, but the nucleic acidmolecule is present extrachromosomally or at a chromosomal location thatis different from its natural chromosomal location.

“Isolated nucleic acid encoding an anti-NSP4 antibody” refers to one ormore nucleic acid molecules encoding antibody heavy and light chains (orfragments thereof), including such nucleic acid molecule(s) in a singlevector or separate vectors, and such nucleic acid molecule(s) present atone or more locations in a host cell.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicaland/or bind the same epitope, except for possible variant antibodies,e.g., containing naturally occurring mutations or arising duringproduction of a monoclonal antibody preparation, such variants generallybeing present in minor amounts. In contrast to polyclonal antibodypreparations, which typically include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody of amonoclonal antibody preparation is directed against a single determinanton an antigen. Thus, the modifier “monoclonal” indicates the characterof the antibody as being obtained from a substantially homogeneouspopulation of antibodies, and is not to be construed as requiringproduction of the antibody by any particular method. For example, themonoclonal antibodies to be used in accordance with the presentinvention may be made by a variety of techniques, including but notlimited to the hybridoma method, recombinant DNA methods, phage-displaymethods, and methods utilizing transgenic animals containing all or partof the human immunoglobulin loci, such methods and other exemplarymethods for making monoclonal antibodies being described herein.

A “naked antibody” refers to an antibody that is not conjugated to aheterologous moiety (e.g., a cytotoxic moiety) or radiolabel. The nakedantibody may be present in a pharmaceutical formulation.

“Native antibodies” refer to naturally occurring immunoglobulinmolecules with varying structures. For example, native IgG antibodiesare heterotetrameric glycoproteins of about 150,000 daltons, composed oftwo identical light chains and two identical heavy chains that aredisulfide-bonded. From N- to C-terminus, each heavy chain has a variableregion (VH), also called a variable heavy domain or a heavy chainvariable domain, followed by three constant domains (CH1, CH2, and CH3).Similarly, from N- to C-terminus, each light chain has a variable region(VL), also called a variable light domain or a light chain variabledomain, followed by a constant light (CL) domain. The light chain of anantibody may be assigned to one of two types, called kappa (κ) andlambda (λ), based on the amino acid sequence of its constant domain.

The term “package insert” is used to refer to instructions customarilyincluded in commercial packages of therapeutic products, that containinformation about the indications, usage, dosage, administration,combination therapy, contraindications and/or warnings concerning theuse of such therapeutic products.

“Percent (%) amino acid sequence identity” with respect to a referencepolypeptide sequence is defined as the percentage of amino acid residuesin a candidate sequence that are identical with the amino acid residuesin the reference polypeptide sequence, after aligning the sequences andintroducing gaps, if necessary, to achieve the maximum percent sequenceidentity, and not considering any conservative substitutions as part ofthe sequence identity. Alignment for purposes of determining percentamino acid sequence identity can be achieved in various ways that arewithin the skill in the art, for instance, using publicly availablecomputer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR)software. Those skilled in the art can determine appropriate parametersfor aligning sequences, including any algorithms needed to achievemaximal alignment over the full length of the sequences being compared.For purposes herein, however, % amino acid sequence identity values aregenerated using the sequence comparison computer program ALIGN-2. TheALIGN-2 sequence comparison computer program was authored by Genentech,Inc., and the source code has been filed with user documentation in theU.S. Copyright Office, Washington D.C., 20559, where it is registeredunder U.S. Copyright Registration No. TXU510087. The ALIGN-2 program ispublicly available from Genentech, Inc., South San Francisco, Calif., ormay be compiled from the source code. The ALIGN-2 program should becompiled for use on a UNIX operating system, including digital UNIXV4.0D. All sequence comparison parameters are set by the ALIGN-2 programand do not vary.

In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:100 times thefraction X/Y where X is the number of amino acid residues scored asidentical matches by the sequence alignment program ALIGN-2 in thatprogram's alignment of A and B, and where Y is the total number of aminoacid residues in B. It will be appreciated that where the length ofamino acid sequence A is not equal to the length of amino acid sequenceB, the % amino acid sequence identity of A to B will not equal the %amino acid sequence identity of B to A. Unless specifically statedotherwise, all % amino acid sequence identity values used herein areobtained as described in the immediately preceding paragraph using theALIGN-2 computer program.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of an activeingredient contained therein to be effective, and which contains noadditional components which are unacceptably toxic to a subject to whichthe formulation would be administered.

A “pharmaceutically acceptable carrier” refers to an ingredient in apharmaceutical formulation, other than an active ingredient, which isnontoxic to a subject. A pharmaceutically acceptable carrier includes,but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. An individual is successfully “treated”, for example, if oneor more symptoms associated with the disorder are mitigated oreliminated. For example, an individual is successfully “treated” if oneor more symptoms associated with an inflammatory disease are mitigatedor eliminated, including, but are not limited to, decreasing symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, delaying the progression of the disease,and/or prolonging survival of individuals

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during or after administration of the other treatment modalityto the individual.

As used herein, the term “prevention” includes providing prophylaxiswith respect to occurrence or recurrence of a disorder in an individual.An individual may be predisposed to a disorder, susceptible to adisorder, or at risk of developing a disorder, but has not yet beendiagnosed with the disorder. In some embodiments, NSP4 inhibitorsdescribed herein are used to delay development of the disorder. In someembodiments, the NSP4 inhibitors described herein prevent inflammationand/or vascular leakage.

As used herein, an individual “at risk” of developing a disorder may ormay not have detectable disease or symptoms of disease, and may or maynot have displayed detectable disease or symptoms of disease prior tothe treatment methods described herein. “At risk” denotes that anindividual has one or more risk factors, which are measurable parametersthat correlate with development of the disorder, as known in the art. Anindividual having one or more of these risk factors has a higherprobability of developing the disorder than an individual without one ormore of these risk factors.

An “effective amount” refers to at least an amount effective, at dosagesand for periods of time necessary, to achieve the desired or indicatedeffect, including a therapeutic or prophylactic result. An effectiveamount can be provided in one or more administrations.

A “therapeutically effective amount” is at least the minimumconcentration required to effect a measurable improvement of aparticular disorder. A therapeutically effective amount herein may varyaccording to factors such as the disease state, age, sex, and weight ofthe patient, and the ability of the antibody to elicit a desiredresponse in the individual. A therapeutically effective amount is alsoone in which any toxic or detrimental effects of the antibody areoutweighed by the therapeutically beneficial effects. A“prophylactically effective amount” refers to an amount effective, atthe dosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically but not necessarily, since a prophylacticdose is used in subjects prior to or at the earlier stage of disease,the prophylactically effective amount can be less than thetherapeutically effective amount.

The term “vector,” as used herein, refers to a nucleic acid moleculecapable of propagating another nucleic acid to which it is linked. Theterm includes the vector as a self-replicating nucleic acid structure aswell as the vector incorporated into the genome of a host cell intowhich it has been introduced. Certain vectors are capable of directingthe expression of nucleic acids to which they are operatively linked.Such vectors are referred to herein as “expression vectors.”

“Antibody-dependent cell-mediated cytotoxicity” or “ADCC” refers to aform of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs)present on certain cytotoxic cells (e.g., natural killer (NK) cells,neutrophils and macrophages) enable these cytotoxic effector cells tobind specifically to an antigen-bearing target cell and subsequentlykill the target cell with cytotoxins. The antibodies “arm” the cytotoxiccells and are required for killing of the target cell by this mechanism.The primary cells for mediating ADCC, NK cells, express FcγRIII only,whereas monocytes express FcγRI, FcγRII and FcγRIII. Fc expression onhematopoietic cells is summarized in Table 3 on page 464 of Ravetch andKinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess ADCC activity ofa molecule of interest, an in vitro ADCC assay, such as that describedin U.S. Pat. No. 5,500,362 or 5,821,337 may be performed. Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and natural killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal., PNAS USA 95:652-656 (1998).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen. To assess complementactivation, a CDC assay, e.g., as described in Gazzano-Santoro et al.,J. Immunol. Methods 202: 163 (1996), may be performed.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

As used herein and in the appended claims, the singular forms “a,” “an,”and “the” include plural reference unless the context clearly indicatesotherwise. For example, reference to an “antibody” is a reference tofrom one to many antibodies, such as molar amounts, and includesequivalents thereof known to those skilled in the art, and so forth.

It is understood that aspect and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

II. Compositions and Methods

A. NSP4 Inhibitors

Provided herein are NSP4 inhibitors which block, inhibit, reduce, orinterfere with the enzymatic activity of a NSP4 in vitro, in situ,and/or in vivo. A NSP4 inhibitor is a molecule having one or more of thefollowing characteristics: (1) inhibits or reduces NSP4 enzymaticactivity; (2) the ability to inhibit or reduce binding of a NSP4 to itssubstrate(s); (3) the ability to increase clearance of a NSP4 (e.g.,decrease extracellular levels of released NSP4); (4) the ability toinhibit or reduce NSP4 release from neutrophils, basophils and/oreosinophils; (5) the ability to reduce NSP4 expression (such as at themRNA level and/or at protein level) in neutrophils, basophils and/oreosinophils; (6) the ability to interact, bind, or recognize a precursorand/or mature form of a NSP4; (7) the ability to enhance inactivation ofa NSP4 by a protease inhibitor (e.g., α1-antitrypsin); (8) the abilityto specifically interact with or bind to a NSP4 and not with neutrophilelastase (NE), cathepsin G (CG), or proteinase 3 (PR3) activity; (9) theability to treat, ameliorate, or prevent any aspect of aneutrophil-mediated disease or disorder described or contemplatedherein; and (10) the ability to treat, ameliorate, or prevent any aspectof a disease or disorder mediated by granulocytes described orcontemplated herein.

Exemplary NSP4 inhibitors that inhibit the production of a NSP4 includeagents such as, but not limited to, compounds that specifically inhibitNSP4 synthesis and/or release, antisense molecules directed to a NSP4,or a short interfering RNA (siRNA) molecule directed to a nucleic acidencoding a NSP4. Additional exemplary NSP4 inhibitors that inhibit NSP4protease activity include agents such as, but not limited to, anti-NSP4antibodies that specifically bind to a NSP4 (e.g., a precursor NSP4and/or a mature NSP4), protease inhibitors (e.g., serine proteaseinhibitors), compounds that specifically inhibit NSP4 catalytic activitysuch as small molecule inhibitors and/or peptide inhibitors, compoundsthat specifically inhibit a NSP4 binding to its substrate(s), a NSP4structural analog, or an RNA or DNA aptamer that binds a NSP4. In someembodiments, a NSP4 inhibitor is an allosteric inhibitor. In someembodiments, a NSP4 inhibitor is an orthosteric inhibitor.

In certain embodiments, a NSP4 inhibitor is a protease inhibitor thatreduces the catalytic activity of a NSP4. In certain embodiments, aprotease inhibitor contemplated herein reduces levels of NSP4 proteaseactivity by reducing active NSP4 protein concentrations. In someembodiments, the NSP4 inhibitor is a serine protease inhibitor, referredto as a serpin, well known in the art as a group of proteins capable ofinhibiting protease activity. For example serpin proteins include, butare not limited to, α1-antitrypsin, C1 inhibitor, heparin-activatedantithrombin, and α2-antiplasmin. Methods for making or purifying serpininhibitors are well known in the art, including, but are not limited to,recombinant protein expression, or immunoaffinity purification.

In certain embodiments, the NSP4 inhibitor is a small molecule inhibitor(e.g., peptide inhibitor), including, but not limited to, small peptidesor peptide-like molecules, soluble peptides, and synthetic non-peptidylorganic or inorganic compounds. A small molecule inhibitor may have amolecular weight of any of about 100 to about 20,000 daltons (Da), about500 to about 15,000 Da, about 1000 to about 10,000 Da. For example,small molecule inhibitors contemplated herein include, but are notlimited to, amastatin hydrochloride hydrate, antipain dihydrochloride,aprotinin, elastatinal, epiamastatin hydrochloride, histatin 5,leupeptin hemisulfate, leupeptin trifluoroacetate, leupeptinhydrochloride, pepstatin, or phenylmethanesulfonylfluoride. In someembodiments, the small molecule inhibitor is a macrocyclic inhibitorsuch as, but not limited to, macrocyclic acylsulfonamides or smallmolecule inhibitors that are known to inhibit the HCV NS3/4A protease.Methods for making and testing the inhibitory effect a small moleculehas on catalytic activity is well known in the art and such methods canbe used to assess the effect of the small molecule inhibitor on NSP4activity. For example, a library of NSP4 inhibitor candidates can bescreened for decreasing NSP4 protease activity by incubating eachinhibitor candidate with active NSP4 in the presence of a fluorogenicpeptide substrate in a buffer. Upon NSP4 cleavage, said fluorogenicpeptide substrate will fluoresce and NSP4 activity in the presence ofeach inhibitor candidate can be measured by techniques well known in theart. Exemplary fluorogenic peptide substrates that can used in assaysdescribed herein include, but are not limited to, a fluorogenic peptidesubstrate with the amino acid sequence ¹IR{Arg(Me)}SSYSFKK¹⁰ or¹IR{Arg}SSYSFKK¹⁰.

In certain embodiments, the NSP4 inhibitor is an anti-NSP4 antibody thatbinds or physically interacts with a NSP4. The antibody may havenanomolar or even picomolar affinities for the target antigen (e.g.,NSP4). In certain embodiments, the Kd of the antibody is about 0.05 toabout 100 nM. For example, Kd of the antibody is any of about 100 nM,about 50 nM, about 10 nM, about 1 nM, about 500 pM, about 100 pM, orabout 50 pM to any of about 2 pM, about 5 pM, about 10 pM, about 15 pM,about 20 pM, or about 40 pM. Methods for the preparation and selectionof antibodies that interact and/or bind with specificity to a NSP4 aredescribed herein.

In certain embodiments, the NSP4 inhibitor comprises at least oneantisense molecule capable of blocking or decreasing the expression of afunctional NSP4 by targeting nucleic acids encoding a NSP4. Nucleic acidsequences of NSP4 are known in the art. For example, a human NSP4 canhave a nucleic acid sequence as shown in NCBI Accession number NM_214710and a mouse NSP4 can have a nucleic acid sequence as shown in NCBIAccession number NM_001042710. Methods are known for the preparation ofantisense oligonucleotide molecules and such methods can be used toprepare antisense oligonucleotides that will specifically bind one ormore of a NSP4 mRNA without cross-reacting with other polynucleotides.Exemplary sites of targeting include, but are not limited to, theinitiation codon, the 5′ regulatory regions, the coding sequence,including any conserved consensus regions, and the 3′ untranslatedregion. In certain embodiments, the antisense oligonucleotides are about10 to about 100 nucleotides in length, about 15 to about 50 nucleotidesin length, about 18 to about 25 nucleotides in length, or more. Incertain embodiments, the oligonucleotides further comprise chemicalmodifications to increase nuclease resistance and the like, such as, forexample, phosphorothioate linkages and 2′-O-sugar modifications known tothose of ordinary skill in the art.

In certain embodiments, the NSP4 inhibitor comprises at least one siRNAmolecule capable of blocking or decreasing the expression of afunctional NSP4 by targeting nucleic acids encoding a NSP4. Methods forpreparation of siRNA molecules are well known in the art and suchmethods can be used to prepare siRNA molecules that will specificallytarget a NSP4 mRNA without cross-reacting with other polynucleotides.siRNA molecules may be generated by methods such as by typical solidphase oligonucleotide synthesis, and often will incorporate chemicalmodifications to increase half-life and/or efficacy of the siRNA agent,and/or to allow for a more robust delivery formulation. Alternatively,siRNA molecules are delivered using a vector encoding an expressioncassette for intracellular transcription of siRNA.

In certain embodiments, the NSP4 inhibitor is an RNA or DNA aptamer thatbinds or physically interacts with a NSP4, and blocks interactionsbetween a NSP4 and its substrate(s). In certain embodiments, the aptamercomprises at least one RNA or DNA aptamer that binds to a mature form ofNSP4. In certain embodiments, the aptamer comprises at least one RNA orDNA aptamer that binds to a precursor form of NSP4.

In certain embodiments, the NSP4 inhibitor comprises at least one NSP4structural analog. The term NSP4 structural analog refers to compoundsthat have a similar three dimensional structure as part of that of aNSP4 (e.g., a precursor and/or mature form of NSP4) and which bind to aNSP4 substrate under physiological conditions in vitro or in vivo,wherein the binding at least partially inhibits a NSP4 biologicalactivity. Suitable NSP4 structural analogs can be designed andsynthesized through molecular modeling of NSP4 binding to its substrate.The NSP4 structural analogs can be monomers, dimers, or higher ordermultimers in any desired combination of the same or different structuresto obtain improved affinities and biological effects. In someembodiments, the NSP4 inhibitor binds to or interacts with an amino acidsequence of a NSP4 as shown in FIG. 1.

Assays

NSP4 inhibitors may be identified and/or characterized using methodswell known in the art, such as, for example, radiolabeled inhibitorassays, optical assays, protein binding assays, biochemical screeningassays, immunoassays, mass shift measurement assays, fluorescenceassays, and/or fluorogenic peptide cleavage assays.

Binding Assays and Other Assays

In certain embodiments, NSP4 inhibitors can be identified by techniqueswell known in the art for detecting the presence of a NSP4 inhibitorcandidate's interaction and/or binding affinity to a NSP4.

In certain embodiments, NSP4 inhibitors that interact with a NSP4 can beidentified using a radiolabeled inhibitor assay. For example, a knownamount of a radiolabeled inhibitor candidate may be incubated with aknown amount of immobilized NSP4 and a buffer. Subsequently, theimmobilized NSP4 may be washed with a buffer and the immobilized NSP4may be measured for the remaining presence of the radiolabeled NSP4inhibitor candidate using techniques known in the art, such as, forexample, a gamma counter. A measurement indicating the presence of aradiolabeled substance may indicate the radiolabeled inhibitor candidateis capable of interacting with and/or binding to NSP4.

In certain embodiments, a NSP4 inhibitor that interacts with a NSP4 maybe identified using an optical technique. An exemplary optical techniqueto detect an inhibitor of a NSP4 may include, e.g., attaching NSP4 to acolorimetric resonant grafting surface, thereby shifting the wavelengthof reflected light due to changes in the optical path the light musttake, and subsequently measuring additional changes in the wavelength ofreflected light when an inhibitor candidate is allowed to interact withNSP4. For example, no change in the measured wavelength of reflectedlight when an inhibitor is incubated with NSP4 may indicate that theinhibitor candidate is unable to interact with NSP4. Changes in themeasured wavelength of reflected light when an inhibitor candidate isincubated with NSP4 may indicate that the inhibitor candidate is capableof binding and/or interacting with NSP4.

In certain embodiments, a NSP4 inhibitor that interacts with a NSP4 maybe identified using a protein binding assay. An exemplary proteinbinding assay to detect a NSP4 inhibitor may include, e.g.,co-immunoprecipitation of a NSP4 in the presence of the inhibitorcandidate. For example, a NSP4 may be incubated with the inhibitorcandidate in buffer, and subsequently an immobilized molecule specificto capture a NSP4, such as, for example, an anti-NSP4 antibody, may beused to capture NSP4 in the presence of the inhibitor candidate and bindthe NSP4, potentially with an interacting inhibitor candidate, duringwash procedures known in the art. Subsequently, NSP4, potentially withan interacting inhibitor candidate, can be released and the presence ofan inhibitor candidate may be detected, based on the inhibitor candidatecharacteristics, by techniques, such as, for example, mass spectrometryand/or Western blot.

In certain embodiments, a NSP4 inhibitor that interacts with a NSP4 maybe identified using a biochemical and/or an immunoassay assay well knownin the art. An exemplary technique may include, e.g., an assay toquantitatively measure changes in NSP4 concentration and/or proteinhalf-life using techniques, such as, for example, Western blot. Forexample, an inhibitor candidate may be incubated with a samplecontaining a NSP4, and subsequently NSP4 protein quantity may bemeasured at points during a time course study. Changes in proteinquantity and/or protein half-life in comparison to a control treatmentmay indicate that the NSP4 inhibitor candidate may be capable ofaltering NSP4 half-life and/or activity.

In certain embodiments, a mass shift measurement assay may be used toidentify a NSP4 inhibitor that interacts with a NSP4. An exemplary massshift measurement assay may include, e.g., detecting the presence of astrongly and/or covalently bound NSP4 inhibitor by measuring a change inNSP4 mass when the inhibitor candidate is interacting with NSP4 by usinginstruments, such as, but not limited to, a mass spectrometer. Forexample, a mass shift assay may be performed on a whole protein and/or apeptide-based analysis, depending on the nature of the inhibitorcandidate interaction. Detection of a mass shift correlating with theaddition of said inhibitor candidate to NSP4 may indicate that theinhibitor candidate may be capable of inhibiting a NSP4. Additionally,an exemplary mass shift measurement assay may include, e.g., detectingthe addition of mass to NSP4 correlating with the respective inhibitorcandidate mass when the inhibitor candidate is interacting with NSP4using techniques, such as, for example, surface plasmon resonance. Forexample, the change in the refractive index of light may be measured andcorrelated with a change in mass of NSP4 attached to a sensor surface.

In certain embodiments, a chemical cross-linking assay may be used toidentify a NSP4 inhibitor that interacts with a NSP4. For example, aninhibitor candidate may be incubated with a NSP4, in vivo or in vitro,with a molecule cross-linker capable of covalently linking an inhibitorcandidate interacting with NSP4 to said NSP4 molecule. Subsequently,techniques, such as, but not limited to, mass spectrometry and/orWestern blot, may be used to identify an inhibitor candidate that may becapable of inhibiting NSP4. For example, detection of NSP4 covalentlycross-linked with the inhibitor candidate may indicate that theinhibitor candidate may be capable of inhibiting NSP4.

In certain embodiments, NSP4 inhibitors that interact with a NSP4 may beidentified using a fluorescence inhibitor assay. For example, a knownamount of a fluorescent inhibitor candidate may be incubated with aknown amount of immobilized NSP4 and a buffer. Subsequently, theimmobilized NSP4 may be washed with a buffer and the immobilized NSP4may be measured for the remaining presence of a fluorescent NSP4inhibitor candidate using techniques known in the art, such as, but notlimited to, fluorescence detection. A measurement indicating thepresence of a fluorescent substance may indicate the fluorescentinhibitor candidate is capable of interacting with and/or binding toNSP4.

Activity Assays

Assays known in the art and described herein (e.g., Example 3) can beused for identifying and testing biological activities of NSP4inhibitors. In some embodiments, assays for testing NSP4 inhibitorsability for blocking a NSP4 activity are provided. An exemplary test forbiological activity may include, e.g., providing a NSP4 (e.g., humanNSP4) in a mixture with a NSP4 inhibitor and incubating the mixture withone or more internally-quenched fluorogenic peptide substrate andmeasuring the fluorescence intensity with an instrument, such as, forexample, a spectrophotometer. An increase in fluorescence in thepresence of a NSP4 inhibitor would indicate the NSP4 inhibitor is unableto block NSP4 activity, while a lack of increase in fluorescence in thepresence of a NSP4 inhibitor would indicate the NSP4 inhibitor blocksNSP4 activity. Exemplary fluorogenic peptide substrates that can used inassays described herein include, but are not limited to, a fluorogenicpeptide substrate with the amino acid sequence ¹IR{Arg(Me)}SSYSFKK¹⁰ or¹IR{Arg}SSYSFKK¹⁰.

In some embodiments, the NSP4 inhibitor may block at least about any of50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% of NSP4 activity in any ofthese assays.

Anti-NSP4 Antibodies

In one aspect, the invention provides isolated antibodies that bind to aNSP4. In certain embodiments, an anti-NSP4 antibody has one or more ofthe following characteristics: (1) binds a NSP4 (e.g., a human NSP4) andinhibits or reduces NSP4 protease activity; (2) blocks binding of a NSP4to its peptide substrate(s); (3) binds to a NSP4 of a mouse and/or ahuman; (4) binds to an inactive and/or mature form of NSP4; (5) binds toa complex formed between a NSP4 and a protease inhibitor (e.g.,α1-antitrypsin); (6) enhances inactivation of a NSP4 by a proteaseinhibitor (e.g., α1-antitrypsin); and (7) reacts specifically with aNSP4 and not with neutrophil elastase (NE), cathepsin G (CG), orproteinase 3 (PR3).

In another aspect, the invention provides an isolated anti-NSP4 antibodythat can be classified into one of the following subclasses: 1) Binds tomature and precursor forms of a mouse NSP4 but not to mature andprecursor forms of a human NSP4; 2) Binds to mature and precursor formsof a human NSP4 but not to mature and precursor forms of a mouse NSP4;3) Binds to a mature form of a mouse NSP4 but not to a precursor form ofa mouse NSP4; 4) Binds to a mature form of a human NSP4 but not to aprecursor form of a human NSP4; 5) Binds to mature forms of a mouse NSP4and a human NSP4 but not to precursor forms of a mouse NSP4 and a humanNSP4; and 6) Binds to mature and precursor forms of a mouse NSP4 and ahuman NSP4.

Described herein is the finding that NSP4 includes an active site and aheparin binding site (see, e.g., FIG. 4A). Further described herein areantibodies that specifically bind to one of these sites (see, e.g.,FIGS. 30-31). In another aspect, the invention provides an isolatedanti-NSP4 antibody that can be classified into one of the followingsubclasses: (1) specifically binds an NSP4 active site or near an NSP4active site; (2) inhibits catalytic activity of NSP4; (3) specificallybinds an NSP4 active site or near an NSP4 active site and inhibitscatalytic activity of NSP4; (4) specifically binds an NSP4 heparinbinding site; (5) competes with heparin for binding to NSP4; and (6)specifically binds an NSP4 heparin binding site and competes withheparin for binding to NSP4.

In one aspect, the invention provides an isolated anti-NSP4 antibodycomprising at least one, two, three, four, five, or six HVRs selectedfrom (i) an HVR-L1 comprising the amino acid sequence of SEQ ID NO:10;(ii) an HVR-L2 comprising the amino acid sequence of SEQ ID NO:11; (iii)an HVR-L3 comprising the amino acid sequence of SEQ ID NO:12; (iv) anHVR-H1 comprising the amino acid sequence of SEQ ID NO:1, 4, or 7; (v)an HVR-H2 comprising the amino acid sequence of SEQ ID NO:2, 5, or 8;and (vi) an HVR-H3 comprising the amino acid sequence of SEQ ID NO:3, 6,or 9.

In one embodiment, the isolated anti-NSP4 antibody comprises a heavychain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3sequence, wherein:

(SEQ ID NO: 1) (a) the HVR-H1 sequence is GFTFSNTYIS; (SEQ ID NO: 2) (b)the HVR-H2 sequence is GFIYPANGATYYADSVKG; and (SEQ ID NO: 3) (c)the HVR-H3 sequence is RRYRLSFDY.

In one embodiment, the isolated anti-NSP4 antibody comprises a heavychain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3sequence, wherein:

(SEQ ID NO: 4) (a) the HVR-H1 sequence is GFTFSGNDIS; (SEQ ID NO: 5) (b)the HVR-H2 sequence is AGISPYGGSTYYADSVKG; and (SEQ ID NO: 6) (c)the HVR-H3 sequence is RRVSFYSRHAGMDY.

In one embodiment, the isolated anti-NSP4 antibody comprises a heavychain variable region comprising an HVR-H1, an HVR-H2, and an HVR-H3sequence, wherein:

(SEQ ID NO: 7) (a) the HVR-H1 sequence is GFTFTSYAIS; (SEQ ID NO: 8) (b)the HVR-H2 sequence is AGISPSNGYTNYADSVKG; and (SEQ ID NO: 9) (c)the HVR-H3 sequence is RAGRWTHSDIDY.

In some embodiments, a heavy chain polypeptide provided herein isfurther combined with a variable region light chain comprising anHVR-L1, an HVR-L2, and an HVR-L3, wherein:

(SEQ ID NO: 10) (a) the HVR-L1 sequence is RASQDVSTAVA; (SEQ ID NO: 11)(b) the HVR-L2 sequence is SASFLYS; and (SEQ ID NO: 12) (c)the HVR-L3 sequence is QQSYTTPPT.

In another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3,wherein:

(SEQ ID NO: 1) (i) the HVR-H1 sequence is GFTFSNTYIS; (SEQ ID NO: 2)(ii) the HVR-H2 sequence is GFIYPANGATYYADSVKG; (SEQ ID NO: 3) (iii)the HVR-H3 sequence is RRYRLSFDY;and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3,wherein:

(SEQ ID NO: 10) (i) the HVR-L1 sequence is RASQDVSTAVA; (SEQ ID NO: 11)(ii) the HVR-L2 sequence is SASFLYS; and (SEQ ID NO: 12) (iii)the HVR-L3 sequence is QQSYTTPPT.

In another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

(c) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3,wherein:

(SEQ ID NO: 4) (i) the HVR-H1 sequence is GFTFSGNDIS; (SEQ ID NO: 5)(ii) the HVR-H2 sequence is AGISPYGGSTYYADSVKG; (SEQ ID NO: 6) (iii)the HVR-H3 sequence is RRVSFYSRHAGMDY;and/or

(d) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3,wherein:

(SEQ ID NO: 10) (i) the HVR-L1 sequence is RASQDVSTAVA; (SEQ ID NO: 11)(ii) the HVR-L2 sequence is SASFLYS; and (SEQ ID NO: 12) (iii)the HVR-L3 sequence is QQSYTTPPT.

In another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

(e) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3,wherein:

(SEQ ID NO: 7) (i) the HVR-H1 sequence is GFTFTSYAIS; (SEQ ID NO: 8)(ii) the HVR-H2 sequence is AGISPSNGYTNYADSVKG; (SEQ ID NO: 9)(iii) the HVR-H3 sequence is RAGRWTHSDIDY;

and/or

(f) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3,wherein:

(SEQ ID NO: 10) (i) the HVR-L1 sequence is RASQDVSTAVA; (SEQ ID NO: 11)(ii) the HVR-L2 sequence is SASFLYS; and (SEQ ID NO: 12)(iii) the HVR-L3 sequence is QQSYTTPPT.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, and IgG4. In a still further specific aspect, the humanconstant region is IgG1. In a still further aspect, the murine constantregion is selected from the group consisting of IgG1, IgG2A, IgG2B, andIgG3. In a still further aspect, the murine constant region is IgG2A. Ina still further specific aspect, the antibody has reduced or minimaleffector function.

In yet another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

-   -   (a) the heavy chain further comprises an HVR-H1, an HVR-H2, and        an HVR-H3 sequence having at least 85% sequence identity to        GFTFSNTYIS (SEQ ID NO:1), GFIYPANGATYYADSVKG (SEQ ID NO:2), and        RRYRLSFDY (SEQ ID NO:3), respectively, and/or    -   (b) the light chain further comprises an HVR-L1, an HVR-L2, and        an HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:10), SASFLYS (SEQ ID NO:11), and        QQSYTTPPT (SEQ ID NO:12), respectively.

In yet another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

-   -   (a) the heavy chain further comprises an HVR-H1, an HVR-H2, and        an HVR-H3 sequence having at least 85% sequence identity to        GFTFSGNDIS (SEQ ID NO:4), AGISPYGGSTYYADSVKG (SEQ ID NO:5), and        RRVSFYSRHAGMDY (SEQ ID NO:6), respectively, and/or    -   (b) the light chain further comprises an HVR-L1, an HVR-L2, and        an HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:10), SASFLYS (SEQ ID NO:11), and        QQSYTTPPT (SEQ ID NO:12), respectively.

In yet another embodiment, provided is an isolated anti-NSP4 antibody orantigen binding fragment comprising a heavy chain variable region and alight chain variable region, wherein:

-   -   (a) the heavy chain further comprises an HVR-H1, an HVR-H2, and        an HVR-H3 sequence having at least 85% sequence identity to        GFTFTSYAIS (SEQ ID NO:7), AGISPSNGYTNYADSVKG (SEQ ID NO:8), and        RAGRWTHSDIDY (SEQ ID NO:9), respectively, and/or    -   (b) the light chain further comprises an HVR-L1, an HVR-L2, and        an HVR-L3 sequence having at least 85% sequence identity to        RASQDVSTAVA (SEQ ID NO:10), SASFLYS (SEQ ID NO:11), and        QQSYTTPPT (SEQ ID NO:12), respectively.

In a still further embodiment, provided is an isolated anti-NSP4antibody or antigen binding fragment comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 13) EVQLVESGGGLVQPGGSLRLSCAASGFTFSNTYISWVRQAPGKGLEWVGFIYPANGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRRY RLSFDYWGQGTLVTVSS,and/or

(b) the light chain sequence has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKR.

In a still another embodiment, provided is an isolated anti-NSP4antibody or antigen binding fragment comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 14) EVQLVESGGGLVQPGGSLRLSCAASGFTFSGNDISWVRQAPGKGLEWVAGISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRRVSFYSRHAGMDYWGQGTLVTVSS,and/or

(b) the light chain sequence has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKR.

In a still another embodiment, provided is an isolated anti-NSP4antibody or antigen binding fragment comprising a heavy chain variableregion and a light chain variable region, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 15) EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYAISWVRQAPGKGLEWVAGISPSNGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRAG RWTHSDIDYWGQGTLVTVSS,and/or

(b) the light chain sequence has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 16) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQ GTKVEIKR.

In any of the above embodiments, an anti-NSP4 antibody is an isolatedantibody. In any of the above embodiments, an anti-NSP4 antibody ishumanized. In one embodiment, an anti-NSP4 antibody comprises HVRs as inany of the above embodiments and HVRs (including HVRs comprising KabatCDR, Chothia CDR, or Contact CDR sequences) shown in FIG. 13, andfurther comprises an acceptor human framework, e.g. a humanimmunoglobulin framework or a human consensus framework.

In certain embodiments, an anti-NSP4 antibody described herein comprisesHVRs as defined by Kabat, e.g., an anti-NSP4 antibody comprising CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs isdefined by Kabat as further described herein. In certain embodiments, ananti-NSP4 antibody described herein comprises HVRs as defined byChothia, e.g., an anti-NSP4 antibody comprising CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs is defined byChothia as further described herein. In certain embodiments, ananti-NSP4 antibody described herein comprises HVRs as defined by ContactCDR sequences, e.g., an anti-NSP4 antibody comprising CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs is definedby Contact CDR sequences as further described herein.

In another aspect, an anti-NSP4 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:16. In certainembodiments, a VL sequence having at least 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-NSP4 antibody comprising that sequenceretains the ability to bind to a NSP4. In certain embodiments, a totalof 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted,inserted and/or deleted in SEQ ID NO:16. In certain embodiments, thesubstitutions, insertions, or deletions occur in regions outside theHVRs (i.e., in the FRs). Optionally, the anti-NSP4 antibody comprisesthe VL sequence of SEQ ID NO:16, including post-translationalmodifications of that sequence.

In another aspect, an anti-NSP4 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence selected from the group consisting of SEQ ID NOS:13-15. Incertain embodiments, a VH sequence having at least 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (e.g.,conservative substitutions), insertions, or deletions relative to thereference sequence, but an anti-NSP4 antibody comprising that sequenceretains the ability to bind to NSP4. In certain embodiments, a total of1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have been substituted,inserted and/or deleted in any of SEQ ID NO: 13-15. In certainembodiments, substitutions, insertions, or deletions occur in regionsoutside the HVRs (i.e., in the FRs). Optionally, the anti-NSP4 antibodycomprises the VH sequence in any of SEQ ID NOS:13-15, includingpost-translational modifications of that sequence.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and RDDVPAVFTSAMDY (SEQ IDNO:52), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSX₁X₂X₃PX₄T (SEQ ID NO:95),wherein X₁ is Y or A; X₂ is T, G, or D; X₃ is T or F; and X₄ is P or L,respectively.

In some embodiments, the anti-NSP4 antibody of the preceding paragraphcomprises a light chain comprising an HVR-L3 sequence having at least85% sequence identity to a sequence selected from SEQ ID NO:12 and92-94.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and (SEQ ID NO:52),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), QQSYTTPPT (SEQ ID NO:12), respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:78 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and RDDVPAVFTSAMDY (SEQ IDNO:52), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYGFPLT (SEQ ID NO:92),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:78 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:102.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and RDDVPAVFTSAMDY (SEQ IDNO:52), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYDFPLT (SEQ ID NO:93),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:78 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:103.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and (SEQ ID NO:52),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSAGFPLT (SEQ ID NO:94),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising the sequence of SEQ ID NO:78 and/or a light chainvariable region comprising the sequence of SEQ ID NO:104.

In some embodiments, provided herein is an anti-NSP4 antibody comprisingan HVR-H2, an HVR-H3, and an HVR-L3 sequence having at least 85%sequence identity to GSISPDNGDTNYADSVKG (SEQ ID NO:51), RDDVPAVFTSAMDY(SEQ ID NO:52), and QQSX₁X₂X₃PX₄T (SEQ ID NO:95), wherein X₁ is Y or A;X₂ is T, G, or D; X₃ is T or F; and X₄ is P or L, respectively.

In some embodiments, the antibody comprises an HVR-H2, an HVR-H3, and anHVR-L3 sequence having at least 85% sequence identity toGSISPDNGDTNYADSVKG (SEQ ID NO:51), RDDVPAVFTSAMDY (SEQ ID NO:52), andQQSYTTPPT (SEQ ID NO:12), respectively. In some embodiments, theantibody comprises an HVR-H2, an HVR-H3, and an HVR-L3 sequence havingat least 85% sequence identity to GSISPDNGDTNYADSVKG (SEQ ID NO:51),RDDVPAVFTSAMDY (SEQ ID NO:52), and QQSYGFPLT (SEQ ID NO:92),respectively. In some embodiments, the antibody comprises an HVR-H2, anHVR-H3, and an HVR-L3 sequence having at least 85% sequence identity toGSISPDNGDTNYADSVKG (SEQ ID NO:51), RDDVPAVFTSAMDY (SEQ ID NO:52), andQQSYDFPLT (SEQ ID NO:93), respectively. In some embodiments, theantibody comprises an HVR-H2, an HVR-H3, and an HVR-L3 sequence havingat least 85% sequence identity to GSISPDNGDTNYADSVKG (SEQ ID NO:51),RDDVPAVFTSAMDY (SEQ ID NO:52), and QQSAGFPLT (SEQ ID NO:94),respectively.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and KRHLHNVAFDY (SEQ IDNO:87), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQAYSAPPT (SEQ ID NO:96),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:105 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:106.

In some embodiments, provided herein is an anti-NSP4 antibody comprisingan HVR-H2, an HVR-H3, and an HVR-L3 sequence having at least 85%sequence identity to AWISPTGGNTYYADSVKG (SEQ ID NO:66), KRHLHNVAFDY (SEQID NO:87), and QQAYSAPPT (SEQ ID NO:96), respectively.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66) or AWIPTAGGNTYYADSVKG (SEQ IDNO:88), and X₁X₂X₃FHNVAFDY (SEQ ID NO:91), wherein X₁ is K or R; X₂ isS, G, or V; and X₃ is L or F, respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQX₁X₂X₃X₄PPT (SEQ ID NO:101),wherein X₁ is S, A, N, or T; X₂ is Y, N, or F; X₃ is T, S, or N; and X₄is T, A, or S, respectively.

In some embodiments, the anti-NSP4 antibody of the preceding paragraphcomprises a heavy chain comprising an HVR-H3 sequence having at least85% sequence identity to a sequence selected from SEQ ID NO:67, 89, and90. In some embodiments, the anti-NSP4 antibody of the precedingparagraph comprises a light chain comprising an HVR-L3 sequence havingat least 85% sequence identity to a sequence selected from SEQ ID NO:12and 97-100.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), KSLFHNVAFDY (SEQ ID NO:67),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:83 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWIPTAGGNTYYADSVKG (SEQ ID NO:88), and KSLFHNVAFDY (SEQ IDNO:67), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTAPPT (SEQ ID NO:97),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:107 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:108.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and KSLFHNVAFDY (SEQ IDNO:67), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQANSTPPT (SEQ ID NO:98),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:83 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:109.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and RGLFHNVAFDY (SEQ IDNO:89), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTAPPT (SEQ ID NO:97),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:110 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:108.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and RVFFHNVAFDY (SEQ IDNO:90), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQNFSSPPT (SEQ ID NO:99),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:111 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:112.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and KSLFHNVAFDY (SEQ IDNO:67), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTAPPT (SEQ ID NO:97),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:83 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:108.

In some embodiments, the antibody comprises a heavy chain and a lightchain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), RGLFHNVAFDY (SEQ ID NO:89),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQTYNAPPT (SEQ ID NO:100),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:110 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:113.

In some embodiments, provided herein is an anti-NSP4 antibody comprisingan HVR-H2, an HVR-H3, and an HVR-L3 sequence having at least 85%sequence identity to AWISPTGGNTYYADSVKG (SEQ ID NO:66) orAWIPTAGGNTYYADSVKG (SEQ ID NO:88), X₁X₂X₃FHNVAFDY (SEQ ID NO:91),wherein X₁ is K or R; X₂ is S, G, or V; and X₃ is L or F; andQQX₁X₂X₃X₄PPT (SEQ ID NO:101), wherein X₁ is S, A, N, or T; X₂ is Y, N,or F; X₃ is T, S, or N; and X₄ is T, A, or S; respectively. In someembodiments, the antibody comprises an HVR-H2, an HVR-H3, and an HVR-L3sequence having at least 85% sequence identity to AWISPTGGNTYYADSVKG(SEQ ID NO:66), KSLFHNVAFDY (SEQ ID NO:67), and QQSYTTPPT (SEQ IDNO:12), respectively. In some embodiments, the antibody comprises anHVR-H2, an HVR-H3, and an HVR-L3 sequence having at least 85% sequenceidentity to AWIPTAGGNTYYADSVKG (SEQ ID NO:88), KSLFHNVAFDY (SEQ IDNO:67), and QQSYTAPPT (SEQ ID NO:97), respectively. In some embodiments,the antibody comprises an HVR-H2, an HVR-H3, and an HVR-L3 sequencehaving at least 85% sequence identity to AWISPTGGNTYYADSVKG (SEQ IDNO:66), KSLFHNVAFDY (SEQ ID NO:67), and QQANSTPPT (SEQ ID NO:98),respectively. In some embodiments, the antibody comprises an HVR-H2, anHVR-H3, and an HVR-L3 sequence having at least 85% sequence identity toAWISPTGGNTYYADSVKG (SEQ ID NO:66), RGLFHNVAFDY (SEQ ID NO:89), andQQSYTAPPT (SEQ ID NO:97), respectively. In some embodiments, theantibody comprises an HVR-H2, an HVR-H3, and an HVR-L3 sequence havingat least 85% sequence identity to AWISPTGGNTYYADSVKG (SEQ ID NO:66),RVFFHNVAFDY (SEQ ID NO:90), and QQNFSSPPT (SEQ ID NO:99), respectively.In some embodiments, the antibody comprises an HVR-H2, an HVR-H3, and anHVR-L3 sequence having at least 85% sequence identity toAWISPTGGNTYYADSVKG (SEQ ID NO:66), KSLFHNVAFDY (SEQ ID NO:67), andQQSYTAPPT (SEQ ID NO:97), respectively. In some embodiments, theantibody comprises an HVR-H2, an HVR-H3, and an HVR-L3 sequence havingat least 85% sequence identity to AWISPTGGNTYYADSVKG (SEQ ID NO:66),RGLFHNVAFDY (SEQ ID NO:89), and QQTYNAPPT (SEQ ID NO:100), respectively.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSWIS (SEQ IDNO:20), GTISPYNGSTYYADSVKG (SEQ ID NO:21), and RVLRPKVYASVMDY (SEQ IDNO:22), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:68 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGYSIH (SEQ IDNO:23), AGISPTNGYTDYADSVKG (SEQ ID NO:24), and RLVFYRGVMDY (SEQ IDNO:25), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:69 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNWIS (SEQ IDNO:26), GYIYPASGYTDYADSVKG (SEQ ID NO:27), and SDSPHAYWYAMDY (SEQ IDNO:28), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:70 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTNNSIS (SEQ IDNO:29), GAISPNNGSTYYADSVKG (SEQ ID NO:30), and RNAWHYSWVGVMDY (SEQ IDNO:31), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:71 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTDYSIH (SEQ IDNO:32), AEIYPYSGDTYYADSVKG (SEQ ID NO:33), and RDGDGWFDWAMDY (SEQ IDNO:34), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:72 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSSTAIS (SEQ IDNO:35), GEIYPSDGYTDYADSVKG (SEQ ID NO:36), and RVKWAVSSLGVMDY (SEQ IDNO:37), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:73 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTDSDIS (SEQ IDNO:38), AWISPSDGATDYADSVKG (SEQ ID NO:39), and HEASDDDYAIDY (SEQ IDNO:40), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

n some embodiments, the antibody comprises a heavy chain variable regioncomprising a sequence having at least 85% sequence identity to SEQ IDNO:74 and/or a light chain variable region comprising a sequence havingat least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDYWIS (SEQ IDNO:41), AGISPNNGDTYYADSVKG (SEQ ID NO:42), and REDDDERDYAMDY (SEQ IDNO:43), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively).

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:75 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTGYGIS (SEQ IDNO:44), GWIYPASGATYYADSVKG (SEQ ID NO:45), and RHRAFDWYPYYIGSSVMDY (SEQID NO:46), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:76 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDYSIS (SEQ IDNO:47), GEINPAGGATYYADSVKG (SEQ ID NO:48), and RGDFPFWSDAYYVMDY (SEQ IDNO:49), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively).

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:77 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSDNDIS (SEQ IDNO:50), GSISPDNGDTNYADSVKG (SEQ ID NO:51), and RDDVPAVFTSAMDY (SEQ IDNO:52), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:78 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSDIS (SEQ IDNO:53), GEIYPSNGDTYYADSVKG (SEQ ID NO:54), and RSVRPSWWAMDY (SEQ IDNO:55), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:79 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSSYDIS (SEQ IDNO:56), GTISPYDGYTDYADSVKG (SEQ ID NO:57), and RYIRRYSVHYGMDY (SEQ IDNO:58), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:80 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTSTSIH (SEQ IDNO:59), AEITPHGGYTNYADSVKG (SEQ ID NO:60), and RGRTKWGWLYGMDY (SEQ IDNO:61), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:81 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTNNSIH (SEQ IDNO:62), AEIAPDDGYTYYADSVKG (SEQ ID NO:63), and RGVIRYAYLYAMDY (SEQ IDNO:64), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:82 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGSGIH (SEQ IDNO:65), AWISPTGGNTYYADSVKG (SEQ ID NO:66), and KSLFHNVAFDY (SEQ IDNO:67), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:83 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSNTYIS (SEQ IDNO:1), GFIYPANGATYYADSVKG (SEQ ID NO:2), and RRYRLSFDY (SEQ ID NO:3),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:84 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFSGNDIS (SEQ IDNO:4), AGISPYGGSTYYADSVKG (SEQ ID NO:5), and RRVSFYSRHAGMDY (SEQ IDNO:6), respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:85 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments, provided herein is an anti-NSP4 antibody comprisinga heavy chain and a light chain, wherein

(a) the heavy chain comprises an HVR-H1, an HVR-H2, and an HVR-H3sequence having at least 85% sequence identity to GFTFTSYAIS (SEQ IDNO:7), AGISPSNGYTNYADSVKG (SEQ ID NO:8), and RAGRWTHSDIDY (SEQ ID NO:9),respectively; and/or

(b) the light chain comprises an HVR-L1, an HVR-L2, and an HVR-L3sequence having at least 85% sequence identity to RASQDVS (SEQ IDNO:19), SASFLYS (SEQ ID NO:11), and QQSYTTPPT (SEQ ID NO:12),respectively.

In some embodiments, the antibody comprises a heavy chain variableregion comprising a sequence having at least 85% sequence identity toSEQ ID NO:86 and/or a light chain variable region comprising a sequencehaving at least 85% sequence identity to SEQ ID NO:16.

In some embodiments that can be combined with any of the embodimentsdescribed above, the sequence identity can be at least 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity.

In some of the above embodiments, an anti-NSP4 antibody is an isolatedantibody. In some of the above embodiments, an anti-NSP4 antibody ishumanized. In one embodiment, an anti-NSP4 antibody comprises HVRs as inany of the above embodiments and HVRs (including HVRs comprising KabatCDR, Chothia CDR, or Contact CDR sequences) of the antibodies describedherein.

In certain embodiments, an anti-NSP4 antibody described herein comprisesHVRs as defined by Kabat, e.g., an anti-NSP4 antibody comprising CDR-H1,CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs isdefined by Kabat as further described herein. In certain embodiments, ananti-NSP4 antibody described herein comprises HVRs as defined byChothia, e.g., an anti-NSP4 antibody comprising CDR-H1, CDR-H2, CDR-H3,CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs is defined byChothia as further described herein. In certain embodiments, ananti-NSP4 antibody described herein comprises HVRs as defined by ContactCDR sequences, e.g., an anti-NSP4 antibody comprising CDR-H1, CDR-H2,CDR-H3, CDR-L1, CDR-L2, and CDR-L3, wherein each of the CDRs is definedby Contact CDR sequences as further described herein.

In another aspect, an anti-NSP4 antibody is provided, wherein theantibody comprises a light chain variable domain (VL) having at least90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequenceidentity to the amino acid sequence selected from SEQ ID NO:16, 102,103, 104, 106, 108, 109, 112, or 113. In certain embodiments, a VLsequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% identity contains substitutions (e.g., conservative substitutions),insertions, or deletions relative to the reference sequence, but ananti-NSP4 antibody comprising that sequence retains the ability to bindto a NSP4. In certain embodiments, a total of 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 amino acids have been substituted, inserted and/or deleted in anyof SEQ ID NO:16, 102, 103, 104, 106, 108, 109, 112, or 113. In certainembodiments, the substitutions, insertions, or deletions occur inregions outside the HVRs (i.e., in the FRs). Optionally, the anti-NSP4antibody comprises the VL sequence of any of SEQ ID NO:16, 102, 103,104, 106, 108, 109, 112, or 113, including post-translationalmodifications of those sequences. Optionally, the light chain contains aconstant region with the sequence of SEQ ID NO:115.

In another aspect, an anti-NSP4 antibody comprises a heavy chainvariable domain (VH) sequence having at least 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acidsequence selected from SEQ ID NO:13-15, 68-86, 105, 107, 110, and 111.In certain embodiments, a VH sequence having at least 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions(e.g., conservative substitutions), insertions, or deletions relative tothe reference sequence, but an anti-NSP4 antibody comprising thatsequence retains the ability to bind to NSP4. In certain embodiments, atotal of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids have beensubstituted, inserted and/or deleted in any of SEQ ID NO:13-15, 68-86,105, 107, 110, and 111. In certain embodiments, substitutions,insertions, or deletions occur in regions outside the HVRs (i.e., in theFRs). Optionally, the anti-NSP4 antibody comprises the VH sequence inany of SEQ ID NO:13-15, 68-86, 105, 107, 110, and 111, includingpost-translational modifications of those sequences. Optionally, theheavy chain contains a constant region with the sequence of SEQ IDNO:114.

In a further aspect of the invention, an anti-NSP4 antibody according toany of the above embodiments is a monoclonal antibody, including achimeric, humanized or human antibody. In one embodiment, an anti-NSP4antibody is an antibody fragment, e.g., a Fv, Fab, Fab′, scFv, diabody,or F(ab′)₂ fragment. In another embodiment, the antibody is a fulllength antibody, e.g., an intact IgG1 antibody or other antibody classor isotype (e.g., IgG₂, IgG₃, or IgG₄) as defined herein.

Amino acid sequences of NSP4 are known in the art. For example, a humanNSP4 can have an amino acid sequence as shown in NCBI Accession numberNP_999875.1, a non-human primate NSP4 can have an amino acid sequence asshown in NCBI Accession number XP_001146596.1 (chimpanzee NSP4),XP_002828403.1 (orangutan NSP4), EHH29398.1 (rhesus monkey NSP4),XP_003914595.1 (baboon NSP4), XP_002761565.1 (marmoset NSP4), orXP_003788878.1 (galago NSP4), a non-primate mammal can have an aminoacid sequence as shown in NCBI Accession number NP_001036175.1 (mouseNSP4), Q6IE59.2 (rat NSP4), XP_004717061.1 (hedgehog NSP4),XP_001375784.7 (opossum NSP4), XP_542217.3 (dog NSP4), XP_593377.3 (cowNSP4) or XP_004009516.1 (sheep NSP4). In some embodiments, the anti-NSP4antibody binds to human NSP4. In some embodiments, the anti-NSP4antibody binds to mouse NSP4. In some embodiments, the anti-NSP4antibody binds to both a human NSP4 and a mouse NSP4. In someembodiments, the anti-NSP4 antibody binds to an amino acid sequence of aNSP4 as shown in FIG. 1.

In a further aspect, an anti-NSP4 antibody according to any of the aboveembodiments may incorporate any of the features, singly or incombination, as described in Sections below:

Antibody Affinity

In certain embodiments, an antibody provided herein has a dissociationconstant (Kd) of ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to 10⁻¹³M, e.g.,from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, Kd is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest (e.g., consistent withassessment of the anti-VEGF antibody, Fab-12, in Presta et al., CancerRes. 57:4593-4599 (1997)). The Fab of interest is then incubatedovernight; however, the incubation may continue for a longer period(e.g., about 65 hours) to ensure that equilibrium is reached.Thereafter, the mixtures are transferred to the capture plate forincubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, Kd is measured using surface plasmonresonance assays using a BIACOR®-2000 or a BIACORE®-3000 (BIAcore, Inc.,Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at ˜10response units (RU). Briefly, carboxymethylated dextran biosensor chips(CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (Kd) is calculated as the ratio k_(off)/k_(on). See, e.g., Chenet al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M⁻¹s⁻¹ by the surface plasmon resonance assay above, then the on-rate canbe determined by using a fluorescent quenching technique that measuresthe increase or decrease in fluorescence emission intensity(excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence ofincreasing concentrations of antigen as measured in a spectrometer, suchas a stop-flow equipped spectrophotometer (Aviv Instruments) or a8000-series SLM-AMINCO spectrophotometer (ThermoSpectronic) with astirred cuvette.

Antibody Fragments

In certain embodiments, an antibody provided herein is an antibodyfragment. Antibody fragments include, but are not limited to, Fab, Fab′,Fab′-SH, F(ab′)₂, Fv, and scFv fragments, and other fragments describedbelow. For a review of certain antibody fragments, see Hudson et al.Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g.,Pluckthün, in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315(1994); see also WO 93/16185; and U.S. Pat. Nos. 5,571,894 and5,587,458. For discussion of Fab and F(ab′)₂ fragments comprisingsalvage receptor binding epitope residues and having increased in vivohalf-life, see U.S. Pat. No. 5,869,046.

Diabodies are antibody fragments with two antigen-binding sites that maybe bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161;Hudson et al., Nat. Med. 9:129-134 (2003); and Hollinger et al., Proc.Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodiesare also described in Hudson et al., Nat. Med. 9:129-134 (2003).

Single-domain antibodies are antibody fragments comprising all or aportion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 B1).

Antibody fragments can be made by various techniques, including but notlimited to proteolytic digestion of an intact antibody as well asproduction by recombinant host cells (e.g. E. coli or phage), asdescribed herein.

Chimeric and Humanized Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

Human Antibodies

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSEtechnology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELOCIMOUSE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbori. J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

Library-Derived Antibodies

Antibodies of the invention may be isolated by screening combinatoriallibraries for antibodies with the desired activity or activities. Forexample, a variety of methods are known in the art for generating phagedisplay libraries and screening such libraries for antibodies possessingthe desired binding characteristics such as the methods described inExample 3. Additional methods are reviewed, e.g., in Hoogenboom et al.in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., HumanPress, Totowa, N.J., 2001) and further described, e.g., in theMcCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marksand Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed.,Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004);Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); andLee et al., J. Immunol. Methods 284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

Multispecific Antibodies

In certain embodiments, an antibody provided herein is a multispecificantibody, e.g. a bispecific antibody. Multispecific antibodies aremonoclonal antibodies that have binding specificities for at least twodifferent sites. In certain embodiments, one of the bindingspecificities is for a NSP4 and the other is for any other antigen. Incertain embodiments, bispecific antibodies may bind to two differentepitopes of a NSP4. Bispecific antibodies can be prepared as full lengthantibodies or antibody fragments.

Techniques for making multispecific antibodies include, but are notlimited to, recombinant co-expression of two immunoglobulin heavychain-light chain pairs having different specificities (see Milstein andCuello, Nature 305: 537 (1983)), WO 93/08829, and Traunecker et al.,EMBO J. 10: 3655 (1991)), and “knob-in-hole” engineering (see, e.g.,U.S. Pat. No. 5,731,168). Multi-specific antibodies may also be made byengineering electrostatic steering effects for making antibodyFc-heterodimeric molecules (WO 2009/089004A1); cross-linking two or moreantibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennanet al., Science, 229: 81 (1985)); using leucine zippers to producebi-specific antibodies (see, e.g., Kostelny et al., J. Immunol.,148(5):1547-1553 (1992)); using “diabody” technology for makingbispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl.Acad. Sci. USA, 90:6444-6448 (1993)); and using single-chain Fv (sFv)dimers (see, e.g. Gruber et al., J. Immunol., 152:5368 (1994)); andpreparing trispecific antibodies as described, e.g., in Tutt et al. J.Immunol. 147: 60 (1991).

Engineered antibodies with three or more functional antigen bindingsites, including “Octopus antibodies,” are also included herein (see,e.g. US 2006/0025576A1).

The antibody or fragment herein also includes a “Dual Acting FAb” or“DAF” comprising an antigen binding site that binds to a NSP4 as well asanother, different antigen (see, US 2008/0069820, for example).

Antibody Variants

In certain embodiments, amino acid sequence variants of the antibodiesprovided herein are contemplated. For example, it may be desirable toimprove the binding affinity and/or other biological properties of theantibody. Amino acid sequence variants of an antibody may be prepared byintroducing appropriate modifications into the nucleotide sequenceencoding the antibody, or by peptide synthesis. Such modificationsinclude, for example, deletions from, and/or insertions into and/orsubstitutions of residues within the amino acid sequences of theantibody. Any combination of deletion, insertion, and substitution canbe made to arrive at the final construct, provided that the finalconstruct possesses the desired characteristics, e.g., antigen-binding.

Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “conservative substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 1 Exemplary Substitutions. Preferred Original Residue ExemplarySubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;

b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;

c. acidic: Asp, Glu;

d. basic: His, Lys, Arg;

e. residues that influence chain orientation: Gly, Pro;

f. aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided comprising an Fcregion wherein a carbohydrate structure attached to the Fc region hasreduced fucose or lacks fucose, which may improve ADCC function.Specifically, antibodies are contemplated herein that have reducedfusose relative to the amount of fucose on the same antibody produced ina wild-type CHO cell. That is, they are characterized by having a loweramount of fucose than they would otherwise have if produced by nativeCHO cells (e.g., a CHO cell that produce a native glycosylation pattern,such as, a CHO cell containing a native FUT8 gene). In certainembodiments, the antibody is one wherein less than about 50%, 40%, 30%,20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. Forexample, the amount of fucose in such an antibody may be from 1% to 80%,from 1% to 65%, from 5% to 65% or from 20% to 40%. In certainembodiments, the antibody is one wherein none of the N-linked glycansthereon comprise fucose, i.e., wherein the antibody is completelywithout fucose, or has no fucose or is afucosylated. The amount offucose is determined by calculating the average amount of fucose withinthe sugar chain at Asn297, relative to the sum of all glycostructuresattached to Asn 297 (e. g. complex, hybrid and high mannose structures)as measured by MALDI-TOF mass spectrometry, as described in WO2008/077546, for example. Asn297 refers to the asparagine residuelocated at about position 297 in the Fc region (Eu numbering of Fcregion residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Suchfucosylation variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana etal.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Antibody variants with at least one galactose residue inthe oligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, the antibody variants comprising an Fc regiondescribed herein are capable of binding to an FcγRIII. In certainembodiments, the antibody variants comprising an Fc region describedherein have ADCC activity in the presence of human effector cells orhave increased ADCC activity in the presence of human effector cellscompared to the otherwise same antibody comprising a human wild-typeIgG1Fc region.

Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half-life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);U.S. Pat. No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assays methods maybe employed (see, for example, ACTI™ non-radioactive cytotoxicity assayfor flow cytometry (CellTechnology, Inc. Mountain View, Calif.; andCytoTox 96® non-radioactive cytotoxicity assay (Promega, Madison, Wis.).Useful effector cells for such assays include peripheral bloodmononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively,or additionally, ADCC activity of the molecule of interest may beassessed in vivo, e.g., in an animal model such as that disclosed inClynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q bindingassays may also be carried out to confirm that the antibody is unable tobind C1q and hence lacks CDC activity. See, e.g., C1q and C3c bindingELISA in WO 2006/029879 and WO 2005/100402. To assess complementactivation, a CDC assay may be performed (see, for example,Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, M. S.et al., Blood 101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie,Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance/halflife determinations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues). In an exemplary embodiment, the anti-NSP4antibody comprising the following amino acid substitutions in its Fcregion: S298A, E333A, and K334A.

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.)). Those antibodies comprise an Fcregion with one or more substitutions therein which improve binding ofthe Fc region to FcRn. Such Fc variants include those with substitutionsat one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305,307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826).

See also Duncan & Winter, Nature 322:738-40 (1988); U.S. Pat. No.5,648,260; U.S. Pat. No. 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

Cysteine Engineered Antibody Variants

In certain embodiments, it may be desirable to create cysteineengineered antibodies, e.g., “thioMAbs,” in which one or more residuesof an antibody are substituted with cysteine residues. In particularembodiments, the substituted residues occur at accessible sites of theantibody. By substituting those residues with cysteine, reactive thiolgroups are thereby positioned at accessible sites of the antibody andmay be used to conjugate the antibody to other moieties, such as drugmoieties or linker-drug moieties, to create an immunoconjugate. Incertain embodiments, any one or more of the following residues may besubstituted with cysteine: V205 (Kabat numbering) of the light chain;A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of theheavy chain Fc region. Cysteine engineered antibodies may be generatedas described, e.g., in U.S. Pat. No. 7,521,541.

Antibody Derivatives

In certain embodiments, an antibody provided herein may be furthermodified to contain additional nonproteinaceous moieties that are knownin the art and readily available. The moieties suitable forderivatization of the antibody include but are not limited to watersoluble polymers. Non-limiting examples of water soluble polymersinclude, but are not limited to, polyethylene glycol (PEG), copolymersof ethylene glycol/propylene glycol, carboxymethylcellulose, dextran,polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane,poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids(either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propropylene glycol homopolymers,prolypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols(e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethyleneglycol propionaldehyde may have advantages in manufacturing due to itsstability in water. The polymer may be of any molecular weight, and maybe branched or unbranched. The number of polymers attached to theantibody may vary, and if more than one polymer are attached, they canbe the same or different molecules. In general, the number and/or typeof polymers used for derivatization can be determined based onconsiderations including, but not limited to, the particular propertiesor functions of the antibody to be improved, whether the antibodyderivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceousmoiety that may be selectively heated by exposure to radiation areprovided. In one embodiment, the nonproteinaceous moiety is a carbonnanotube (Kam et al., Proc. Natl. Acad. Sci. USA 102: 11600-11605(2005)). The radiation may be of any wavelength, and includes, but isnot limited to, wavelengths that do not harm ordinary cells, but whichheat the nonproteinaceous moiety to a temperature at which cellsproximal to the antibody-nonproteinaceous moiety are killed.

Recombinant Methods and Compositions

Antibodies may be produced using recombinant methods and compositions,e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment,isolated nucleic acid encoding an anti-NSP4 antibody described herein isprovided. Such nucleic acid may encode an amino acid sequence comprisingthe VL and/or an amino acid sequence comprising the VH of the antibody(e.g., the light and/or heavy chains of the antibody). In a furtherembodiment, one or more vectors (e.g., expression vectors) comprisingsuch nucleic acid are provided. In a further embodiment, a host cellcomprising such nucleic acid is provided. In one such embodiment, a hostcell comprises (e.g., has been transformed with): (1) a vectorcomprising a nucleic acid that encodes an amino acid sequence comprisingthe VL of the antibody and an amino acid sequence comprising the VH ofthe antibody, or (2) a first vector comprising a nucleic acid thatencodes an amino acid sequence comprising the VL of the antibody and asecond vector comprising a nucleic acid that encodes an amino acidsequence comprising the VH of the antibody. In one embodiment, the hostcell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO) cell or lymphoidcell (e.g., Y0, NS0, Sp20 cell). In one embodiment, a method of makingan anti-NSP4 antibody is provided, wherein the method comprisesculturing a host cell comprising a nucleic acid encoding the antibody,as provided above, under conditions suitable for expression of theantibody, and optionally recovering the antibody from the host cell (orhost cell culture medium). Further provided herein are anti-NSP4antibodies produced by such methods.

For recombinant production of an anti-NSP4 antibody, nucleic acidencoding an antibody, e.g., as described above, is isolated and insertedinto one or more vectors for further cloning and/or expression in a hostcell. Such nucleic acid may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody).

Suitable host cells for cloning or expression of antibody-encodingvectors include prokaryotic or eukaryotic cells described herein. Forexample, antibodies may be produced in bacteria, in particular whenglycosylation and Fc effector function are not needed. For expression ofantibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat.Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J., 2003), pp. 245-254, describing expression of antibody fragments inE. coli.) After expression, the antibody may be isolated from thebacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors, including fungi and yeast strains whoseglycosylation pathways have been “humanized,” resulting in theproduction of an antibody with a partially or fully human glycosylationpattern. See Gerngross, Nat. Biotech. 22:1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains have been identified which may be used inconjunction with insect cells, particularly for transfection ofSpodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat.Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429(describing PLANTIBODIES™ technology for producing antibodies intransgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian celllines that are adapted to grow in suspension may be useful. Otherexamples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7); human embryonic kidney line (293 or 293cells as described, e.g., in Graham et al., J. Gen Virol. 36:59 (1977));baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells asdescribed, e.g., in Mather, Biol. Reprod. 23:243-251 (1980)); monkeykidney cells (CV1); African green monkey kidney cells (VERO-76); humancervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo ratliver cells (BRL 3A); human lung cells (W138); human liver cells (HepG2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., inMather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; andFS4 cells. Other useful mammalian host cell lines include Chinesehamster ovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al.,Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines suchas Y0, NS0 and Sp2/0. For a review of certain mammalian host cell linessuitable for antibody production, see, e.g., Yazaki and Wu, Methods inMolecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa,N.J.), pp. 255-268 (2003).

Assays

Anti-NSP4 antibodies provided herein may be identified, screened for, orcharacterized for their physical/chemical properties and/or biologicalactivities by various assays known in the art.

Binding Assays and Other Assays

In one aspect, an antibody of the invention is tested for its antigenbinding activity, e.g., by known methods such as ELISA, Western blot,etc.

In another aspect, competition assays may be used to identify anantibody that competes with one or more antibodies selected from 1-1,1-2, 1-3, 1-5, 2-1, 2-2, 2-3, 2-4, 2-5, 3-2, 3-5, 4-2, 4-3, 4-4, 4-5,5-1, 5-2, 5-3, 5-4, 35.WT, 35.14, 35.50, 35.62, 35.77, 51.WT, 51.30,51.50, 51.51, 51.59, 51.72, and 51.82 for binding to a NSP4. In certainembodiments, such a competing antibody binds to the same epitope (e.g.,a linear or a conformational epitope) that is bound by one or moreantibodies selected from 1-1, 1-2, 1-3, 1-5, 2-1, 2-2, 2-3, 2-4, 2-5,3-2, 3-5, 4-2, 4-3, 4-4, 4-5, 5-1, 5-2, 5-3, 5-4, 35.WT, 35.14, 35.50,35.62, 35.77, 51.WT, 51.30, 51.50, 51.51, 51.59, 51.72, and 51.82.Detailed exemplary methods for mapping an epitope to which an antibodybinds are provided in Morris (1996) “Epitope Mapping Protocols,” inMethods in Molecular Biology vol. 66 (Humana Press, Totowa, N.J.).

In an exemplary competition assay, immobilized NSP4 is incubated in asolution comprising a first labeled antibody that binds to a NSP4 (e.g.,a human NSP4 or a mouse NSP4) and a second unlabeled antibody that isbeing tested for its ability to compete with the first antibody forbinding to NSP4. The second antibody may be present in a hybridomasupernatant. As a control, immobilized NSP4 is incubated in a solutioncomprising the first labeled antibody but not the second unlabeledantibody. After incubation under conditions permissive for binding ofthe first antibody to NSP4, excess unbound antibody is removed, and theamount of label associated with immobilized NSP4 is measured. If theamount of label associated with immobilized NSP4 is substantiallyreduced in the test sample relative to the control sample, then thatindicates that the second antibody is competing with the first antibodyfor binding to NSP4. See Harlow and Lane (1988) Antibodies: A LaboratoryManual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

In another aspect, biolayer interferometry may be used to determine theaffinity of anti-NSP4 antibodies against a NSP4. In an exemplary assay,an anti-NSP4 antibody is immobilized onto anti-human Fc sensors, andincubated with increasing concentrations of NSP4 to obtain affinitymeasurements using an instrument such as, for example, the Octet System(ForteBio).

In another aspect, ELISA may be used to identify an antibody that bindsto a complex of a NSP4 and a protease inhibitor (e.g., α1-antitrypsin).In an exemplary assay, recombinant NSP4 is mixed with a proteaseinhibitor, such as a serine protease inhibitor, to allow complexformation. The NSP4 complexes are coated in nickel (Ni) plates via aHis-tag present on the recombinant NSP4 and a capture ELISA is performedas previously described (see Kuhl et al., 2010, J. Immunol., 185,387-399) with the anti-NSP4 antibody. Binding of anti-NSP4 antibody tothe NSP4 complexes as compared to an isotype antibody control identifiesthe antibody as binding to a complex of NSP4 and a protease inhibitor.See, e.g., Hinkofer et al., J. Biol. Chem., 2013, 288:26635-26648.

In any of the embodiments herein, the NSP4 used in the assay can be amature form or a precursor form of a NSP4.

Activity Assays

Assays known in the art and described herein (e.g., Example 3) can beused for identifying and testing biological activities of anti-NSP4antibodies. In some embodiments, assays for testing anti-NSP4 antibodiesfor blocking NSP4 activity are provided. An exemplary test forbiological activity may include, e.g., providing a NSP4 (e.g., a humanNSP4) in a mixture with an anti-NSP4 antibody and incubating the mixturewith one or more internally-quenched fluorogenic peptide substrate andmeasuring the fluorescence intensity with an instrument, such as, forexample, a spectrophotometer. An increase in fluorescence in thepresence of an anti-NSP4 antibody would indicate the anti-NSP4 antibodyis unable to block NSP4 activity, while a lack of increase influorescence in the presence of an anti-NSP4 antibody would indicate theanti-NSP4 antibody blocks NSP4 activity. Exemplary fluorogenic peptidesubstrates that can used in assays described herein include, but are notlimited to, a fluorogenic peptide substrate with the amino acid sequence¹IR{Arg(Me)}SSYSFKK¹⁰ or ₁IR{Arg}SSYSFKK¹⁰.

In some embodiments, the anti-NSP4 antibody may block at least about anyof 50%, 60%, 70%, 80%, 85%, 90%, 95% and 100% of NSP4 activity in any ofthese assays.

Assays for testing anti-NSP4 antibodies for blocking NSP4 activity arealso provided. An exemplary method for assessing NSP4 activity mayinclude providing granulocytes on a substrate, incubating thegranulocytes with a NSP4 inhibitor, such as an anti-NSP4 antibody,followed by stimulation with a chemotactic factor (such as ComplementC5a or interleukin-8), and measuring a change in chemotaxis or motilityof the granulocytes in the presence of an anti-NSP4 antibody as comparedto an isotype control, wherein a reduction in chemotaxis or motilityindicates the anti-NSP4 antibody is blocking NSP4 activity.

In another exemplary assay, an in vivo animal model for aneutrophil-mediated disease can be used. For example, theneutrophil-dependent K/B×N serum transfer arthritis model can be used byadministering an anti-NSP4 antibody prior to, concomitant with, or afteradministration of intravenous K/B×N serum. Vascular leakage, erythema,and edema in the paws of treated mice can be monitored using in vivonear-infrared fluorescence imaging as described in Example 2, wherein areduction of any one of vascular leakage, erythema, or edema indicatesthe anti-NSP4 antibody blocks NSP4 activity.

Methods and Compositions for Diagnostics and Detection

In certain embodiments, any of the anti-NSP4 antibodies provided hereinis useful for detecting the presence of a NSP4 protein in a biologicalsample. The term “detecting” as used herein encompasses quantitative orqualitative detection. In certain embodiments, a biological samplecomprises a cell or tissue, such as neutrophils.

In one embodiment, an anti-NSP4 antibody for use in a method ofdiagnosis or detection is provided. In a further aspect, a method ofdetecting the presence of a NSP4 in a biological sample is provided. Incertain embodiments, the method comprises contacting the biologicalsample with an anti-NSP4 antibody as described herein under conditionspermissive for binding of the anti-NSP4 antibody to a NSP4, anddetecting whether a complex is formed between the anti-NSP4 antibody anda NSP4. Such method may be an in vitro or in vivo method. In oneembodiment, an anti-NSP4 antibody is used to select subjects eligiblefor therapy with an anti-NSP4 antibody, e.g. where a NSP4 is a biomarkerfor selection of patients.

Exemplary disorders that may be diagnosed using an antibody of theinvention include stroke, diabetic retinopathy, edema, diabetic macularedema, hereditary angioedema, idiopathic angioedema, leakage ofvasculature, cerebral ischemia, acute lung injury, asthma, chronicobstructive pulmonary disease (COPD), acute respiratory distresssyndrome (ARDS), osteoarthritis, rheumatoid arthritis (e.g., juvenilerheumatoid arthritis), septic shock, chronic bronchitis, pulmonaryemphysema, α-1 anti-trypsin deficiency, cystic fibrosis, idiopathicpulmonary fibrosis, systemic lupus erythematosus (SLE), autoimmunevasculitides, blistering skin diseases, cancer (e.g., lung cancer), adisease caused by a deficiency in a natural protease inhibitor (e.g., aserine protease inhibitor), and any other granulocyte-mediated diseaseor disorder (e.g., neutrophil-mediated disease or disorder) describedand contemplated herein.

In certain embodiments, labeled anti-NSP4 antibodies are provided.Labels include, but are not limited to, labels or moieties that aredetected directly (such as fluorescent, chromophoric, electron-dense,chemiluminescent, and radioactive labels), as well as moieties, such asenzymes or ligands, that are detected indirectly, e.g., through anenzymatic reaction or molecular interaction. Exemplary labels include,but are not limited to, the radioisotopes ³²P, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I,fluorophores such as rare earth chelates or fluorescein and itsderivatives, rhodamine and its derivatives, dansyl, umbelliferone,luceriferases, e.g., firefly luciferase and bacterial luciferase (U.S.Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones,horseradish peroxidase (HRP), alkaline phosphatase, (3-galactosidase,glucoamylase, lysozyme, saccharide oxidases, e.g., glucose oxidase,galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclicoxidases such as uricase and xanthine oxidase, coupled with an enzymethat employs hydrogen peroxide to oxidize a dye precursor such as HRP,lactoperoxidase, or microperoxidase, biotin/avidin, spin labels,bacteriophage labels, stable free radicals, and the like.

B. Pharmaceutical Compositions and Formulations

Also provided herein are pharmaceutical compositions and formulationscomprising a NSP4 inhibitor described herein and a pharmaceuticallyacceptable carrier. In some embodiments, the NSP4 inhibitor may be anantibody described herein.

Pharmaceutical compositions and formulations as described herein can beprepared by mixing the NSP4 inhibitor (such as an antibody or apolypeptide) having the desired degree of purity with one or moreoptional pharmaceutically acceptable carriers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions. Pharmaceuticallyacceptable carriers are generally nontoxic to recipients at the dosagesand concentrations employed, and include, but are not limited to:buffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride; benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as polyethylene glycol (PEG). Exemplarypharmaceutically acceptable carriers herein further includeinsterstitial drug dispersion agents such as soluble neutral-activehyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, BaxterInternational, Inc.). Certain exemplary sHASEGPs and methods of use,including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The composition and formulation herein may also contain more than oneactive ingredients as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generallysterile. Sterility may be readily accomplished, e.g., by filtrationthrough sterile filtration membranes.

C. Therapeutic Methods

Any of the NSP4 inhibitors (e.g., an anti-NSP4 antibody) provided hereinmay be used in therapeutic methods.

In one aspect, a NSP4 inhibitor (e.g., an anti-NSP4 antibody) for use asa medicament is provided. In further aspects, a NSP4 inhibitor (e.g., ananti-NSP4 antibody) for use in treating or preventing a disease ordisorder mediated by granulocytes is provided. In certain embodiments, aNSP4 inhibitor (e.g., an anti-NSP4 antibody) for use in a method oftreatment or prevention is provided. In certain embodiments, theinvention provides a NSP4 inhibitor (e.g., an anti-NSP4 antibody) foruse in a method of treating or preventing an individual having a diseaseor disorder mediated by granulocytes comprising administering to theindividual an effective amount of the NSP4 inhibitor. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. Insome embodiments, the disease or disorder mediated by granulocytes isselected from the group consisting of vascular disease and inflammatorydisease. An “individual” according to any of the above embodiments ispreferably a human. In any of the embodiments herein, the disease ordisorder mediated by granulocytes can be a disease described herein.

In certain embodiments, the invention provides an anti-NSP4 antibody foruse in a method of treating or preventing an individual having a diseaseor disorder mediated by granulocytes comprising administering to theindividual an effective amount of the anti-NSP4 antibody. In certainembodiments, the antibody specifically binds an NSP4 active site. Incertain embodiments, the antibody inhibits catalytic activity of NSP4.In certain embodiments, the antibody specifically binds an NSP4 activesite and inhibits catalytic activity of NSP4. In certain embodiments,the antibody specifically binds an NSP4 heparin binding site. In certainembodiments, the antibody competes with heparin for binding to NSP4. Incertain embodiments, the antibody specifically binds an NSP4 heparinbinding site and competes with heparin for binding to NSP4. In certainembodiments, an effective amount of an antibody that specifically bindsan NSP4 active site and/or inhibits catalytic activity of NSP4 and anantibody that specifically binds an NSP4 heparin binding site and/orcompetes with heparin for binding to NSP4 is administered to theindividual.

In a further aspect, the invention provides for the use of a NSP4inhibitor (e.g., an anti-NSP4 antibody) in the manufacture orpreparation of a medicament. In one embodiment, the medicament is fortreatment or prevention of a disease or disorder mediated bygranulocytes. In a further embodiment, the medicament is for use in amethod of treating or preventing a disease or disorder mediated bygranulocytes comprising administering to an individual having thedisease or disorder an effective amount of the medicament. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. Insome embodiments, the disease or disorder mediated by granulocytes isselected from the group consisting of vascular disease and inflammatorydisease. An “individual” according to any of the above embodiments maybe a human. In any of the embodiments herein, the disease or disordermediated by granulocytes can be a disease described herein.

In a further aspect, the invention provides a method for treating orpreventing a disease or disorder mediated by granulocytes. In oneembodiment, the method comprises administering to an individual havingsuch disease or disorder an effective amount of a NSP4 inhibitor (e.g.,an anti-NSP4 antibody). In one such embodiment, the method furthercomprises administering to the individual an effective amount of atleast one additional therapeutic agent. In some embodiments, the diseaseor disorder mediated by granulocytes is selected from the groupconsisting of vascular disease and inflammatory disease. An “individual”according to any of the above embodiments may be a human. In any of theembodiments herein, the disease or disorder mediated by granulocytes canbe a disease described herein.

In one aspect, a NSP4 inhibitor (e.g., an anti-NSP4 antibody) for use asa medicament is provided. In further aspects, a NSP4 inhibitor (e.g., ananti-NSP4 antibody) for use in treating neutrophil-mediated disease ordisorder is provided. In certain embodiments, a NSP4 inhibitor (e.g., ananti-NSP4 antibody) for use in a method of treatment is provided. Incertain embodiments, the invention provides a NSP4 inhibitor (e.g., ananti-NSP4 antibody) for use in a method of treating an individual havinga neutrophil-mediated disease or disorder comprising administering tothe individual an effective amount of the NSP4 inhibitor. In one suchembodiment, the method further comprises administering to the individualan effective amount of at least one additional therapeutic agent. Insome embodiments, the neutrophil-mediated disease or disorder isselected from the group consisting of vascular disease and inflammatorydisease. An “individual” according to any of the above embodiments ispreferably a human. In any of the embodiments herein, theneutrophil-mediated disease or disorder can be a disease describedherein.

In a further aspect, the invention provides for the use of a NSP4inhibitor (e.g., an anti-NSP4 antibody) in the manufacture orpreparation of a medicament. In one embodiment, the medicament is fortreatment of a neutrophil-mediated disease or disorder. In a furtherembodiment, the medicament is for use in a method of treating aneutrophil-mediated disease or disorder comprising administering to anindividual having the disease or disorder an effective amount of themedicament. In one such embodiment, the method further comprisesadministering to the individual an effective amount of at least oneadditional therapeutic agent. In some embodiments, theneutrophil-mediated disease or disorder is selected from the groupconsisting of vascular disease and inflammatory disease. An “individual”according to any of the above embodiments may be a human. In any of theembodiments herein, the neutrophil-mediated disease or disorder can be adisease described herein.

In a further aspect, the invention provides a method for treating aneutrophil-mediated disease or disorder. In one embodiment, the methodcomprises administering to an individual having such disease or disorderan effective amount of a NSP4 inhibitor (e.g., an anti-NSP4 antibody).In one such embodiment, the method further comprises administering tothe individual an effective amount of at least one additionaltherapeutic agent. In some embodiments, the neutrophil-mediated diseaseor disorder is selected from the group consisting of vascular diseaseand inflammatory disease. An “individual” according to any of the aboveembodiments may be a human. In any of the embodiments herein, theneutrophil-mediated disease or disorder can be a disease describedherein.

Neutrophils, also known as polymorphonuclear leukocytes (PMN), are cellsof the innate immune system that are involved in acute inflammation andphagocytosis of invading pathogens (e.g., bacteria). The anti-microbialactivity of neutrophils is partially mediated by neutrophil serineproteases, such as neutrophil elastase, cathepsin G, and proteinase 3,which act intracellularly to destroy phagocytosed microorganisms or canbe released by neutrophils to act extracellularly to contain and reducepathogen proliferation at sites of infection. While neutrophil serineproteases that are released by neutrophils can serve a beneficial rolein the innate immune system, these proteases may also contribute to theformation of neutrophil-mediated disease or disorders by causing, forexample, aberrant tissue damage and inflammation. Studies have linkedneutrophils as playing a contributory role in the pathology of severaldiseases such as, but not limited to, septic shock, acute respiratorydistress syndrome, post-ischemic reperfusion, chronic obstructivepulmonary disease (COPD), rheumatoid arthritis, and cancer. See, e.g.,Adams et al., J Trauma-Injury Infect Crit Care., 2001, 51:452-456;Lindsey et al., Circulation, 2001, 103:2181-2187; Wright et al.,Rheumatology, 2010, 49(9):1618-1631; Magrone et al., Pharm Des., 2012,18(12):1609-19; and Vaguliene et al., BMC Immunology., 2013, 6:14-36.

Neutrophil-mediated diseases or disorders include diseases characterizedby inflammation (e.g., acute inflammation and/or chronic inflammation),increased vascular permeability, tissue damage, and/or otherinflammatory processes in which neutrophils are known to play a role. Insome embodiments, a neutrophil-mediated disease or disorder is avascular disease selected from the group consisting of stroke, diabeticretinopathy, edema, diabetic macular edema, hereditary angioedema,idiopathic angioedema, leakage of vasculature, and cerebral ischemia. Insome embodiments, a neutrophil-mediated disease or disorder is aninflammatory disease selected from the group consisting of acute lunginjury, asthma, chronic obstructive pulmonary disease (COPD), acuterespiratory distress syndrome (ARDS), osteoarthritis, rheumatoidarthritis (e.g., juvenile rheumatoid arthritis), and septic shock. Insome embodiments, a neutrophil-mediated disease or disorder is apulmonary disease selected from the group consisting of COPD, chronicbronchitis, pulmonary emphysema, α-1 anti-trypsin deficiency, cysticfibrosis, idiopathic pulmonary fibrosis, and ARDS. In some embodiments,a neutrophil-mediated disease or disorder is an autoimmune disease suchas, but not limited to, systemic lupus erythematosus (SLE), autoimmunevasculitides, and blistering skin diseases. In some embodiments, theneutrophil-mediated disease or disorder is a cancer such as, but notlimited to, lung cancer, breast cancer, colon cancer, lymphoma,pancreatic cancer, and brain cancer. In some embodiments, the cancer ismetastatic cancer. In some embodiments, a neutrophil-mediated disease ordisorder is a disease caused by a deficiency in a natural (i.e., host)protease inhibitor (e.g., a serine protease inhibitor) such as, but notlimited to, α-1 anti-trypsin, antithrombin, C1 inhibitor, secretoryleukocyte protease inhibitor, monocyte-neutrophil elastase inhibitor,and α1-antichymotrypsin.

Granulocytes include neutrophils, eosinophils, and basophils. In someembodiments, the methods or medicaments described herein are useful fortreating or preventing a disease or disorder mediated by granulocytes.For example, the disease or disorder that can be treated or preventedincludes vascular diseases, inflammatory diseases, and autoimmunediseases. In some embodiments, the disease or disorder is selected fromthe group consisting of stroke, diabetic retinopathy, edema, diabeticmacular edema, hereditary angioedema, idiopathic angioedema, leakage ofvasculature, cerebral ischemia, acute lung injury, anaphylaxis, systemicanaphylaxis, allergic lung inflammation, asthma (e.g., allergic asthma,virus-induced asthma), chronic obstructive pulmonary disease (COPD),acute respiratory distress syndrome (ARDS), idiopathic pulmonaryfibrosis, systemic lupus erythematosus (SLE), autoimmune vasculitides,blistering skin diseases (e.g., bullous pemphigoid), inflammatory skindiseases (e.g., atopic dermatitis, urticarial, eosinophilic cellutitis),cancer (e.g., lung cancer), kidney diseases (e.g., glomerulonephritis),osteoarthritis, rheumatoid arthritis, psoriatic arthritis, psoriasis,septic shock, inflammatory bowel disease (e.g., ulcerative colitis,Crohn's disease).

In a further aspect, the invention provides pharmaceutical compositionsor formulations comprising any of the NSP4 inhibitors provided herein,e.g., for use in any of the above therapeutic methods. In oneembodiment, a pharmaceutical composition or formulation comprises any ofthe NSP4 inhibitors (e.g., anti-NSP4 antibodies) provided herein and apharmaceutically acceptable carrier. In another embodiment, apharmaceutical composition or formulation comprises any of the NSP4inhibitors provided herein and at least one additional therapeuticagent, e.g., as described below.

NSP4 inhibitors described herein can be used either alone or incombination with other agents in a therapy. For instance, a NSP4inhibitor described herein may be co-administered with at least oneadditional therapeutic agent. Such combination therapies encompasscombined administration (where two or more therapeutic agents areincluded in the same or separate formulations), and separateadministration, in which case, administration of the antibody of theinvention can occur prior to, simultaneously, and/or following,administration of the additional therapeutic agent and/or adjuvant.

A NSP4 inhibitor described herein (and any additional therapeutic agent)can be administered by any suitable means, including parenteral,intrapulmonary, and intranasal, and, if desired for local treatment,intralesional administration. Parenteral infusions includeintramuscular, intravenous, intraarterial, intraperitoneal, orsubcutaneous administration. Dosing can be by any suitable route, e.g.by injections, such as intravenous or subcutaneous injections, dependingin part on whether the administration is brief or chronic. Variousdosing schedules including but not limited to single or multipleadministrations over various time-points, bolus administration, andpulse infusion are contemplated herein.

NSP4 inhibitors (e.g., antibodies) described herein would be formulated,dosed, and administered in a fashion consistent with good medicalpractice. Factors for consideration in this context include theparticular disorder being treated, the particular mammal being treated,the clinical condition of the individual patient, the cause of thedisorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The NSP4 inhibitor need not be, but isoptionally formulated with one or more agents currently used to preventor treat the disorder in question. The effective amount of such otheragents depends on the amount of NSP4 inhibitor present in theformulation, the type of disorder or treatment, and other factorsdiscussed above. These are generally used in the same dosages and withadministration routes as described herein, or about from 1 to 99% of thedosages described herein, or in any dosage and by any route that isempirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of aNSP4 inhibitor (e.g., an anti-NSP4 antibody) (when used alone or incombination with one or more other additional therapeutic agents) willdepend on the type of disease to be treated, the type of NSP4 inhibitor,the severity and course of the disease, whether the NSP4 inhibitor isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the treatment, and thediscretion of the attending physician. The NSP4 inhibitor is suitablyadministered to the patient at one time or over a series of treatments.

For example, depending on the type and severity of the disease, about 1μg/kg to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of a NSP4 inhibitor (e.g.,an anti-NSP4 antibody) can be an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. One typical dailydosage might range from about 1 μg/kg to 100 mg/kg or more, depending onthe factors mentioned above. For repeated administrations over severaldays or longer, depending on the condition, the treatment wouldgenerally be sustained until a desired suppression of disease symptomsoccurs. One exemplary dosage of the NSP4 inhibitor (e.g., an anti-NSP4antibody) would be in the range from about 0.05 mg/kg to about 10 mg/kg.Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10mg/kg (or any combination thereof) may be administered to the patient.Such doses may be administered intermittently, e.g. every week or everythree weeks (e.g. such that the patient receives from about two to abouttwenty, or e.g. about six doses of the antibody). An initial higherloading dose, followed by one or more lower doses may be administered.However, other dosage regimens may be useful. The progress of thistherapy is easily monitored by conventional techniques and assays.

III. Articles of Manufacture and Kits

In another aspect of the invention, an article of manufacture or a kitcomprising one or more of the NSP4 inhibitors (e.g., anti-NSP4antibodies) useful for the treatment, prevention and/or diagnosis of thedisorders described above is provided. The article of manufacture or kitmay further comprise a container and a label or package insert on orassociated with the container. Suitable containers include, for example,bottles, vials, syringes, IV solution bags, etc. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition which is by itself or combined withanother composition effective for treating, preventing and/or diagnosingthe condition and may have a sterile access port (for example thecontainer may be an intravenous solution bag or a vial having a stopperpierceable by a hypodermic injection needle). At least one active agentin the composition is a NSP4 inhibitor described herein. The label orpackage insert indicates that the composition is used for treating thecondition of choice. In some embodiments, the condition of choice is adisease or disorder mediated by granulocytes. In some embodiments, thedisease or disorder to be treated is selected from the group consistingof stroke, diabetic retinopathy, edema, diabetic macular edema,hereditary angioedema, idiopathic angioedema, leakage of vasculature,cerebral ischemia, acute lung injury, anaphylaxis, systemic anaphylaxis,allergic lung inflammation, asthma (e.g., allergic asthma, virus-inducedasthma), chronic obstructive pulmonary disease (COPD), acute respiratorydistress syndrome (ARDS), idiopathic pulmonary fibrosis, systemic lupuserythematosus (SLE), autoimmune vasculitides, blistering skin diseases(e.g., bullous pemphigoid), inflammatory skin diseases (e.g., atopicdermatitis, urticarial, eosinophilic cellutitis), cancer (e.g., lungcancer), kidney diseases (e.g., glomerulonephritis), osteoarthritis,rheumatoid arthritis, psoriatic arthritis, psoriasis, septic shock,inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease).In some embodiments, the condition of choice is a neutrophil-mediateddisease or disorder such as, but not limited to, stroke, diabeticretinopathy, edema, diabetic macular edema, hereditary angioedema,idiopathic angioedema, leakage of vasculature, cerebral ischemia, acutelung injury, asthma, chronic obstructive pulmonary disease (COPD), acuterespiratory distress syndrome (ARDS), osteoarthritis, rheumatoidarthritis (e.g., juvenile rheumatoid arthritis), septic shock, chronicbronchitis, pulmonary emphysema, α-1 anti-trypsin deficiency, cysticfibrosis, idiopathic pulmonary fibrosis, systemic lupus erythematosus(SLE), autoimmune vasculitides, blistering skin diseases, cancer (e.g.,lung cancer), a disease caused by a deficiency in a natural proteaseinhibitor (e.g., a serine protease inhibitor), or any otherneutrophil-mediated disease or disorder described and contemplatedherein. Moreover, the article of manufacture or kit may comprise (a) afirst container with a composition contained therein, wherein thecomposition comprises a NSP4 inhibitor described herein; and (b) asecond container with a composition contained therein, wherein thecomposition comprises a second therapeutic agent. The article ofmanufacture or kit in this embodiment of the invention may furthercomprise a package insert indicating that the compositions can be usedto treat a particular condition. Alternatively, or additionally, thearticle of manufacture or kit may further comprise a second (or third)container comprising a pharmaceutically-acceptable buffer, such asbacteriostatic water for injection (BWFI), phosphate-buffered saline,Ringer's solution and dextrose solution. It may further include othermaterials desirable from a commercial and user standpoint, includingother buffers, diluents, filters, needles, and syringes.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention.

The following are examples of methods and compositions of the invention.It is understood that various other embodiments may be practiced, giventhe general description provided above.

EXAMPLES Example 1: Characterization of Substrate Recognition byNeutrophil Serine Protease 4 (NSP4)

NSP4, a member of a family of neutrophil serine proteases characterizedas trypsin-fold proteases, is stored in neutrophil azurophilic granules(Perera et al., J Immunol., 2013), yet it is the least abundant of allNSPs (Perera et al., Proc Natl Acad Sci USA, 2012, 109:6229-6234) andits function remains unknown. NSP4 is highly conserved from bony fish tohuman (FIG. 1) and predates the evolutionary emergence of other NSPs(Perera et al., Proc Natl Acad Sci USA, 2012, 109:6229-6234; Perera etal., Expert Rev Clin Immunol, 2012, 8:501-503). NSP4, therefore, likelyplays fundamental roles in neutrophil biology. While the relativelybroad substrate specificity of neutrophil elastase (NE), cathepsin G(CG), and proteinase 3 (PR3) is well understood based on knowledge oftheir active site structures (Navia et al., Proc Natl Acad Sci USA,1989, 86:7-11; Hof et al., EMBO J, 1996, 15:5481-5491; Fujinaga et al.,J Mol Biol, 1996, 261:267-278), NSP4 poses a challenge in that itcleaves substrates after arginine residues (Perera et al., Proc NatlAcad Sci USA, 2012, 109:6229-6234; Perera et al., J Immunol., 2013), butits primary sequence predicts a different elastase-like active site(FIG. 2A). Among trypsin-fold proteases, the active sites of trypsin,chymotrypsin, and elastase help define the three major classes ofsubstrate specificities at the P1 position (Hedstrom et al, Chem Rev,2002, 102:4501-4524) (FIG. 2B). Trypsin-like proteases, such ascoagulation and complement factors, have a deep S1 pocket to accommodatethe long arginine side chain that is stabilized by a salt bridgeinteraction with the highly conserved D189 (chymotrypsinogen numbering).In contrast, elastase-like proteases cannot accommodate a large arginineside chain owing to their shallow S1 pocket.

To investigate how NSP4 is able to achieve its arginine-specificity withan apparent elastase-like S1 pocket (FIGS. 2A and B), the atypical S1pocket was characterized.

Methods

Recombinant Protein Expression and Purification

To produce human wild-type NSP4 for structural characterization, the DNAencoding human NSP4 from Ile34 (Ile16 in chymotrypsinogen numbering) toAla283 was fused with an N-terminal His₆-tag and enteropeptidasecleavage site and cloned into pAcGP67 vector (BD Biosciences). Theresulting baculovirus transfer vector was confirmed by DNA sequencingand co-transfected with BaculoGold linearized DNA (BD Biosciences) intoSf9 insect cells, and amplified three times to generate a high titerviral stock. For protein production, cells were cultured in shake flasksor in wave bags (GE Healthcare) at 27° C. with ESF921 medium (ExpressionSystems) for 72 hours post-infection and removed by centrifugation. Theresulting supernatant was supplemented with 1 mM NiCl₂, 5 mM CaCl₂, in50 mM Tris-HCl, pH 7.5. The protein in the supernatant was captured on aNi-NTA column (Qiagen) by gravity flow, washed with 200 ml wash buffer(50 mM Tris pH 7.5, 300 mM NaCl, 10 mM imidazole), and eluted withelution buffer (50 mM Tris pH 7.5, 300 mM NaCl, 300 mM imidazole).Protein was concentrated and further purified on a size exclusion column(Superdex 200 HiLoad 16/60, GE Biosciences) equilibrated with 20 mM TrispH 7.5, 150 mM NaCl.

To produce NSP4 mutants for biochemical characterization, the DNAencoding the human NSP4 fragment from Ile34 to Ala283 was cloned with anN-terminal FLAG tag and C-terminal His₆ tag in a modified pRK5 vectorsuitable for mammalian cell expression. The QuikChange mutagenesis kit(Agilent) was used to generate derivative expression constructsincorporating the desired single and double point mutants (inchymotrypsin numbering): F190A, S192A, S195A, S216G, F190A:S216G, andS192A:S216G. The constructs were expressed in Chinese Hamster Ovarian(CHO) cells by transient expression and after incubation the supernatantwas harvested by centrifugation. The protein in the supernatant wascaptured on an anti-FLAG affinity resin by gravity flow, washed with 200ml phosphate-buffered saline (PBS), eluted with elution buffer (50 mMsodium citrate pH 3.0, 150 mM NaCl), and immediately neutralized to pH 7using 1M Tris pH 8.

Biochemical Assays

Both insect cell-derived and CHO cell-derived NSP4 were activated usingenteropeptidase (Invitrogen) at 15 EU/ml of protein, and incubatedovernight at 20° C. Completion of the cleavage reaction was monitored bySDS-PAGE and Liquid chromatography-mass spectrometry (LC-MS). Asecondary purification with ion exchange was performed to removeenteropeptidase and purify NSP4 to homogeneity with a MonoS column (GEHealthcare) using a gradient elution with 20 mM Tris pH 7.5 from 0.05 Mto 1.0 M NaCl.

Crystallization and Structure Determination

For apo-NSP4 and FFR:NSP4 crystals, insect cell-derived NSP4 waspartially deglycosylated using Endo F3 at 1:100 Endo F3:NSP4 mass ratioin 100 mM sodium citrate pH 5.5, 300 mM NaCl for 3 hours at 37° C. andthen overnight at 4° C. The deglycosylation reaction was verified usingSDS-PAGE and LC-MS. For VLK:NSP4 crystals, the insect cell-derived NSP4were fully glycosylated. To make FFR:NSP4 and VLK:NSP4 complexes, NSP4was mixed with D-Phe-L-Phe-L-Arg-cmk (Bachem) or withD-Val-L-Leu-L-Lys-cmk (Bachem), respectively, at 20-fold molar excessfor overnight at 20° C. The resulting FFR:NSP4 and VLK:NSP4 covalentcomplexes were verified using LC-MS. Apo-NSP4, FFR:NSP4, and VLK:NSP4were all desalted using Superdex 5200 HiLoad 16/60 size exclusionchromatography column equilibrated using 20 mM Tris pH 7.5, 150 mM NaCl.The protein was concentrated to approximately 10 mg/ml and mixed 1:1with the precipitant/buffer solution (mother liquor) for crystallizationtrials.

All crystals were obtained by sitting drop vapor diffusion method at 19°C., with the crystals appearing between 2 to 7 days. FFR:NSP4crystallized in 20% PEG MME 2000, 0.1M Tris pH 8.5, 0.2M trimethylamineN-oxide. Apo-NSP4 (form 1) crystallized in 15-17% PEG-10,000, 0.1 Msodium acetate pH 4.4, 0.1 M ammonium acetate. Apo-NSP4 (form 2)crystallized in 22-25% PEG-3350, 0.1 M Bis-Tris pH 5.5, 0.2 M NaCl.VLK:NSP4 crystallized in 20% PEG-3350 0.2 M potassium acetate, with noadditional buffer. The crystals were cryoprotected in mother liquorsupplemented with 20% glycerol and flash frozen in liquid nitrogen.

Data collection experiments were performed using a synchrotron lightsource. Specifically, Apo (forms 1 and 2) and VLK-cmk datasets werecollected at ALS beamline 5.0.1 at the wavelength of 0.9774 Å andtemperature of 95 K (Advanced Light Source). The FFR-cmk dataset wascollected at SSRL beamline 7-1 at the wavelength of 1.1271 Å andtemperature of 100 K (Stanford Synchroton Radiation Light Source). Datawas indexed and integrated using HKL2000 (Otwinowski et al., Methods inEnzymology, 1997, 276:307-326). The initial structure of NSP4 was solvedusing MrBUMP (Keegan et al., Acta Crystallogr D Biol Crystallogr, 2007,63:447-457) to gain initial phases and rebuilt into maps created bysimulated annealing using the PHENIX package (Adams et al., ActaCrystallogr D Biol Crystallogr, 2010, 66:213-221). The structures wererefined using PHENIX with 97.0% (apo form 1), 95.4% (apo form 2), 96.5%(FFR-cmk), and 95.4% (VLK-cmk) of residues in the favored region of theRamachandran plot and the rest in the allowed region. Data collectionand refinement statistics are summarized in Table 2. All structuralfigures were generated using PyMol (Schrödinger, LLC).

Fluorogenic Peptide Cleavage Assay

Internally-quenched fluorogenic peptides with7-methoxycourmain-4-acetate (Mca) and mini-PEG1(8-amino-3,6-dioxaoctanoic acid) at the N-terminus and dinitrophenol(Dnp) attached to the penultimate lysine side chain near the C-terminuswere synthesized using Fmoc solid-phase peptide synthesis (GenScript).The peptides were added to 50 nM NSP4 or 100 nM Factor Xa (EnzymeResearch Laboratories) in 50 mM Tris pH 8.0, 150 mM NaCl, 2 mM CaCl₂ at37° C. Enzyme kinetic measurements were done using SpectraMax M5(MolecularDevices) with the excitation at 328 nm and emission at 393 nm.Kinetic data were fitted using Prism5 (GraphPad Software) using thestandard Michaelis-Menten kinetics model and a standard curve wasgenerated to convert the rate of catalysis from the fluorescencemeasurements (RFU/s) to nanomolar products formed per second (nM/s).LC-MS was used to confirm the site of peptide cleavage using fluorogenicpeptides that were incubated with 50 nM NSP4 or 200 nM Factor Xa in 50mM Tris pH 8.0, 150 mM NaCl, 2 mM CaCl₂ at 37° C. for 1 hour. Thepeptide fragments were separated on a 5-60% acetonitrile:waterreverse-phase gradient before analysis on a time-of-flight massspectrometer (Agilent).

Heparin Binding Assays

Heparin binding was assessed using fluorescence polarization, wherefluorescein-conjugated heparin (Invitrogen) was mixed with purifiedrecombinant NSP4. Fluorescence polarization assays were performed inthree independent measurements and read on a Victor 3 (Perkin Elmer)equipped with 485±30 nm excitation and 535±40 nm emission filters. Toassess the reactions via electrophoretic mobility shift assay, an equalvolume of 50% glycerol was added to NSP4:heparin mixture, which werethen separated by polyacrylamide gel electrophoresis and fluorescencesignal detected with a blue (488 nm) laser and a 526±20 nm emissionfilter using a Typhoon imager (GE Healthcare).

Characterization of NSP4^(−/−) Mice

NSP4^(−/−) mice were generated as previously described (Tang et al., NatBiotechnol, 2010, 28:749-755) and backcrossed to C57/BL6 for more than10 generations. Confirmation of NSP4 ablation and determination ofneighboring protease gene expression in NSP4^(−/−) mice were done usingRT-PCR. RNA from total bone marrow cells of NSP4^(−/−) mice or wild-typelittermates were isolated using the RNeasy Mini kit (Qiagen) and thecorresponding cDNA were synthesized using the iScript reversetranscriptase (Bio-Rad), all performed according to manufacturerinstructions. qPCR were performed from cDNA samples using the TaqMan2×PCR master mix (Applied Biosystems) with the following TaqManprimer/probe sets were obtained from Applied Biosystems, with thecatalogue number in parenthesis: Prss57_1 (ABI Mm01144794_m1, spansexons 1-2), Prss57_2 (ABI Mm01144795_m1, spans exons 2-3), Prss57_3 (ABIMm01144796_m1, spans exons 3-4), Cfd (ABI Mm01143935_g1, spans exons4-5), Elane (ABI Mm01168928_g1, spans exons 1-2), Gzmm (ABIMm00493150_m1, spans exons 2-3), Prtn3 (ABI Mm00478323_m1, spans exons1-2), 18s rRNA (ABI 4333760F).

To quantify immune cell populations, cells from the femoral bone marrowof NSP4^(−/−) mice or wild-type littermates were identified usingstandard flow cytometry protocols and the following antibody clones:anti-CD11b (M1/70); anti-CD11c (N418); anti-B220 (RA3-6B2); anti-Ly6C(HK1.4), anti-Ly6G (1A8). All antibodies were obtained from eBioscience.Viability was evaluated using Sytox Blue (Invitrogen). Stained cellswere sorted using a FACSAria cell sorter (BD Biosciences). The followingpopulations were sorted and counted: B cells, B220⁺; total myeloidcells, B220⁻/CD11b⁺; monocytes, B220⁻/CD11b⁺/Ly6C^(hi), and neutrophils,B220⁻/CD11b⁺/Ly6G^(hi).

K/B×N Serum-Transfer Mouse Model

K/B×N serum-induced vascular permeability was monitored in vivo bynon-invasive near-infrared fluorescence imaging (NIRF) of the mousewhole-paw. Mice were anesthetized by 2% isoflurane (Butler Schein, 1L/min flow), implanted with a tail vein catheter, and immobilized withthe paw secured by surgical tape on the imaging surface glass of theKodak In-Vivo FX Pro 400 whole-animal NIRF imaging system (CarestreamHealth). Mice were injected through the tail vein catheter with 100 μlof the blood pool probe AngioSense 680 (PerkinElmer), imaged at 1 minuteintervals (650 nm excitation/700 nm emission, 21.4 mm FOV, 10 secondexposure, 2× binning) for 5 min to establish baseline fluorescencebefore tail vein catheter injection of 75 ul K/B×N serum and furtherimaging for another 25 min. The K/B×N serum used in this model wassourced from KRN×NOD F1 mice that exhibited severe arthritis. Theaverage fluorescence intensities and the fold change in fluorescenceintensities from the initial imaging time point within paws werequantified using custom routines in MatLab (MathWorks).

Arthritis was assessed daily for 8 days following vascular permeabilityanalysis. Clinical scores range from 0 to 16 per animal, as assigned bycombining the individual paw scores of 0 (normal joint appearance) to 4(maximal erythema and edema). The following joints were analyzed forerythema and edema: tarsal or carpal joints, metatarsal or metacarpaljoints, metatarsalphalangeal or metacarpalphalangeal joints, orphalanges.

Histological Examination of Mouse Arthritic Paws

Paws were fixed in neutral buffered formalin, decalcified and processedroutinely to sagittal hemisections stained with hematoxylin and eosin.All 4 paws per animal were examined. Histological lesions were scored onan arbitrary scale from 0 (normal) to 5 (severe) for the followingfeatures: infiltration with inflammatory cells, fibroplasia includingpannus formation, cartilage injury, and bone remodeling.

Results

Kinetic Characterization of NSP4 S1 Binding Pocket

The ability of NSP4 to recognize a P1-arginine residue was investigated,particularly in light of the F190 and S216 residues in the NSP4 S1binding pocket that appear poised to obstruct arginine binding (FIG.2A). The NSP4 residues F190 and S216, situated on opposite sides of theopening to the S1 pocket, might act as a movable gate to allow access tothe S1 pocket. If so, the P1-arginine side chain could be stabilized byD226 as a surrogate for D189 (G189 in NSP4) (FIG. 2A) (13, 14). To testthis hypothesis, NSP4 variants were engineered having either a partiallyopen (F190A or S216G) or fully open (F190A:S216G) “gate”, all of whichshould increase enzyme activity if the hypothesis was correct. Tomeasure effects on NSP4 activity, a fluorogenic peptide was synthesizedthat was specifically cleaved by NSP4 after arginine as the P1 residue¹IRR³↓⁴SSYSFKK¹⁰ (FIG. 2C). The results showed that the single mutants(F190A or S216G) and the double mutant F190A:S216G had approximately10-fold and 4-fold reduced activity (k_(cat)/K_(M)) compared towild-type (FIG. 2D). These results did not support the movable gatehypothesis, but rather suggested that both F190 and S216 were criticallyimportant for positioning the substrate to enable catalysis.

Crystal Structures of NSP4

To elucidate the structural basis of the P1-arginine recognitionmechanism, the X-ray crystal structure of NSP4 was determined with thecovalently bound substrate mimic D-Phe-L-Phe-L-Arg chloromethyl ketone(FFR-cmk) at 1.40 Å resolution. In addition, two non-isomorphousstructures of NSP4 in its apo-form at 2.55 Å and 2.70 Å resolutions(Table 2) were determined. All structures exhibited the double β-barreland catalytic triad arrangement characteristic of trypsin-fold serineproteases (FIG. 3A). NSP4 also had an extended basic surface patch thatmay function to localize NSP4 to proteoglycans (FIG. 4A). Consistentwith this view, NSP4 exhibited strong heparin binding in fluorescencepolarization and electrophoretic mobility shift assays (FIG. 4B).Superposition of the NSP4 structures also showed that the 99-loop (FIG.3A), which formed the S2/S4 substrate interaction sites, was labile andmight be implicated in NSP4 allosteric regulation as demonstrated forother serine proteases (Ganesan et al., Structure, 2009, 17:1614-1624;Debela et al., J Mol Biol, 2007, 373:1017-1031).

TABLE 2 Data and refinement characteristics. Apo (form 1) Apo (form 2)FFR-CMK VLK-cmk Data Collection Space group P4₁ P6₃ P2₁2₁2₁ P6₅ CellDimensions a, b, c ({acute over (Å)}) 70.4, 70.4, 150.0 86.6, 86.6, 69.455.0, 64.5, 68.4 89.7, 89.7, 108.6 α, β, γ (°) 90, 90, 90 90, 90, 12090, 90, 90 90, 90, 120 Resolution ({acute over (Å)})   50-2.55(2.64-2.55)   50-2.70 (2.80-2.70) 35.69-1.40 (1.45-1.40)  50.0-3.08(3.19-3.08) R_(symm) 0.082 (0.494) 0.149 (0.683) 0.048 (0.765) 0.154(0.810) <I/σI> 14.1 (2.0)  12.8 (2.7)  34.3 (2.8)  12.5 (2.4) Completeness (%) 99.3 (100)  99.8 (100)  93.8 (98.2) 99.2 (99.8)Redundancy 3.8 (3.8) 6.2 (6.3) 7.0 (7.1) 6.6 (6.6) Reflections 8959350790 319648 60991 measured Unique reflections 23577 8192 45664 9241Wilson B ({acute over (Å)}²) 62 52 18 81 X-ray source ALS 5.0.1 ALS5.0.1 SSRL 7-1 ALS 5.0.1 Data reduction HKL2000 HKL2000 HKL2000 HKL2000Refinement Resolution ({acute over (Å)}) 50-2.55 50-2.70 50-1.40 50-3.08Reflections (total) 23565 8192 45530 9240 Reflections in R_(Free) 1203802 2276 423 Molecules per 2 1 1 2 asymmetric unit R_(work) 0.197 0.1870.192 0.185 R_(free) 0.231 0.248 0.215 0.240 Mean B-factor 54 33 20 62Number TLS groups 8 4 4 8 No. atoms 3775 1850 2136 3710 Protein 35721795 1811 3562 Carbohydrate 76 38 38 98 Ligand 0 0 34 50 Solvent 127 17253 0 R.m.s. deviations Bond lengths ({acute over (Å)}) 0.009 0.0080.008 0.009 Bond angles (°) 1.05 1.05 1.30 1.23 Ramachandran (%) 97.095.4 96.5 95.4 Preferred region *Values in parentheses are forhighest-resolution shell.Structural Basis of NSP4 Arginine Specificity

The structural details of the active site revealed an unprecedentedmechanism by which the substrate P1-arginine was recognized andconcurrently explained the NSP4 conundrum. The structures show that thecanonical S1 pocket was non-existent, as it is completely occluded byF190 and S216 (FIG. 3B and FIG. 5). The F190 and S216 side chains werehighly ordered, consistent with the absence of a ‘gate’ mechanism;instead, residues F190 and S216 formed the floor of a shallow grooveabove the occluded S1 pocket to accommodate the redirected arginine sidechain (FIG. 3B). The arginine side chain movement from the canonical“down” to the new “up” position in NSP4 was accomplished by a rotationof the Cβ-Cγ bond (Chi2 angle) by 160° (FIG. 6A). Otherwise, thecatalytic triad, the oxyanion hole, and the classical Cα trace of theFFR peptide, including the antiparallel main chain interactions betweenthe protease 214-216 residues and the substrate P1-P3 residues, were allpreserved. The “up” conformation of P1-arginine was supported by F190,which provided a hydrophobic platform that interacted favorably with thealiphatic portion of the P1-arginine side chain. In addition, thespecificity for P1-arginine was conferred by a network of H-bondsinvolving the guanidinium group, coordinated by three H-bondacceptors—the S216 and S192 side chains and the G217 backbone carbonyloxygen (FIG. 6B). Therefore, NSP4 replaced the predominantP1-arginine-stabilizing electrostatic interaction inside a typicaltrypsin-like S1 pocket with a new H-bond network situated on top of theoccluded S1 pocket. The importance of this H-bond network for catalysiswas examined by mutating the two H-bond acceptors S192 and S216. Thesingle H-bond acceptor mutants showed 10-fold reduced activity, whereasremoval of both H-bond acceptors (S192A:S216G) resulted in 20-foldreduction (FIG. 6C).

Despite NSP4's poor activity towards P1-lysine (P1-Lys) substrates, itwas possible to prepare a complex of NSP4 with the covalently boundlysine substrate mimic D-Val-L-Leu-L-Lys (VLK)-cmk and obtain a crystalstructure at 3.08 Å resolution. It showed that the P1-lysine side chainadopted a conformation like that of P1-arginine (FIG. 7). However,unlike the longer P1-arginine side chain, the shorter P1-lysine sidechain could not engage the full complement of H-bonds as theP1-arginine, thereby explaining the extremely poor cleavage afterP1-lysine residues (FIG. 2C).

NSP4 Processed Substrates with Modified Arginines

In an orthogonal approach to validate the unique NSP4 active site,NSP4's activity towards peptide substrate with modified P1 arginine wasexamined. The naturally occurring arginine-derivative methylarginine isresistant to cleavage by trypsin-like proteases because themethylarginine side chain cannot be spatially accommodated inside thenarrow S1 pocket (Baldwin et al., Science, 1971, 171:579-581; Asami etal., Bioorg Med Chem Lett, 2012, 22:6328-6332). This structurallimitation did not apply to NSP4. Because the P1-arginine guanidiniumgroup was solvent-exposed on NSP4, it was reasoned that NSP4 couldaccommodate modifications to the arginine guanidinium group such as anextra methyl group. Enzymatic assays with peptidic substratesdemonstrated that NSP4 could indeed cleave after methylarginine, whereasthe trypsin-like protease, factor Xa, with a canonical S1 pocket couldonly cleave unmodified P1-Arg substrate (FIG. 6D and FIG. 8).

Example 2: Determining the Biological Role of NSP4 Using NSP4-DeficientMice

As a first step towards exploring the in vivo function of NSP4, thepossible role of NSP4 in inflammation using the neutrophil-dependentK/B×N serum transfer arthritis model was investigated (Monach et al.,Curr Protoc Immunol, 2008, Chapter 15:Unit 15.22). NSPs have beenpreviously implicated in inflammatory arthritis, but required thecombined deficiency of both NE and CG to achieve full protection inexperimental arthritis models, suggesting functional redundancy amongNSPs. However, given the unique arginine specificity of NSP4 and itsevolutionary status, it was surmised that NSP4 might have essentialfunctions in neutrophil-mediated inflammatory processes. To investigatethe role of NSP4 in inflammation, NSP4-deficient mice were generated aspreviously described (Tang et al., Nat Biotechnol, 2010, 28:749-755)(FIG. 9A) and backcrossed to the C57BL/6 strain. Successful ablation ofNSP4 in NSP4^(−/−) mice was verified by RT-qPCR using different PCRprimer/probe sets spanning three different NSP4 exon boundaries (FIG.9B). In a similar manner, NSP4^(−/−) mice were also analyzed for theexpression of protease genes flanking NSP4 in this protease-rich locus.None of the neighboring protease genes, including the NSP members NE andPR3, were affected (FIG. 9B). The NSP4-deficient mice were viable andfertile, exhibited no notable abnormalities in comprehensive phenotypicscreens (Henrich et al., Nat Struct Biol, 2003, 10:520-526), and hadnormal bone marrow cell, B cell, monocyte myeloid cell, and neutrophilcounts (FIG. 9C).

One major hallmark of the K/B×N model is the rapid onset of localizedvascular edema in the mouse paws following intravenous K/B×N serumadministration (Binstadt et al., Nat Immunol, 2006, 7:284-292). Tomonitor vascular leakage, a near-infrared fluorescence vascular probewas administered to both NSP4-deficient and wild-type mice 5 minutesprior to K/B×N serum injection and then visualized and quantified thesubsequent vascular permeability changes by in vivo near-infraredfluorescence imaging (FIGS. 10A and B). Following intravenousadministration of K/B×N serum, the wild-type mice, as expected,exhibited rapid extravasation of the vascular probe in the paws withreproducible severity and kinetics. In striking contrast, thefluorescence levels of NSP4^(−/−) mice remained at baseline throughoutthe study (FIGS. 10A and B), suggesting that NSP4 played anindispensable role in mediating this immune complex-triggeredvasopermeability response.

The initial induction of vascular leakage after K/B×N serum transfer inwild-type mice was followed by the development of erythema and edema,whereas NSP4^(−/−) mice remained significantly protected during the8-day post-induction period (FIG. 11A). Histological examination of thepaws at the experimental end point showed mild to moderate polyarthritisin the wild-type control group while NSP4^(−/−) mice were essentiallydevoid of lesions (FIGS. 11B and 11C). In the wild type mice, thepolyarthritis observed was consistent with the K/B×N model and wascharacterized by infiltration with inflammatory cells, predominantlyneutrophils, accompanied by mild fibroplasia, cartilage injury and boneremodeling (FIG. 11C). In contrast, the NSP4^(−/−) mice had minimalpathological lesions (FIG. 11C, middle right), and virtually noinflammatory cell infiltration (FIG. 11C, bottom right). Taken together,these results indicated NSP4 played an indispensable pro-inflammatoryrole in this neutrophil-mediated disease model.

Example 3: Generation of Conformation-Specific and Species-Specific NSP4Antibodies

A panel of antibodies were generated and characterized for specificityagainst NSP4. Residues were numbered using the Kabat system (Kabat etal., Sequences of proteins of immunological interest, 5th Ed., PublicHealth Service, National Institutes of Health, Bethesda, Md. (1991)).

Methods

Generation of Recombinant Human and Mouse NSP4

Recombinant human NSP4 and mouse NSP4 were generated as antigens forlibrary sorting. NSP4 was produced as an inactive zymogen by engineeringa cleavable epitope tag at the protein N-terminus to prevent prematureactivation and thus reduce cellular toxicity and increase proteinexpression in heterologous expression systems. A total of four NSP4antigens were generated: human zymogen (hZ), human active (hA), mousezymogen (mZ), and mouse active (mA). The hZ NSP4 was expressed in insectcells and purified by N-terminal 6His-tag using immobilized metalaffinity chromatography. The mZ NSP4 was expressed in CHO cells andpurified by N-terminal FLAG tag using anti-FLAG affinity chromatography.Both the hZ and mZ NSP4s were further purified using Superdex 200 sizeexclusion chromatography (GE Healthcare) and MonoS cation exchangechromatography (GE Healthcare). The catalytically active forms of theseNSP4 (hA and mA) were generated by treating the purified zymogen forms(hZ and mZ) with EKmax enteropeptidase (Invitrogen) to remove theN-terminal epitope tag and were subsequently purified over another MonoScation exchange chromatography (GE Healthcare) to homogeneity. Cleavageoccurred on the C-terminal side of lysine following the amino acidsequence Asp-Asp-Asp-Asp-Lys. These four NSP4 antigens (hZ, hA, mZ, andmA) were used in parallel for phage library sorting and for screeningconformational-specific and species-specific anti-NSP4 antibodies (FIG.12).

Phagemid Vectors for Library Construction

Phage-displayed synthetic antibody libraries were generated usingoligonucleotide-directed mutagenesis in the three heavy chaincomplementarity-determining regions (CDRs) on a modified h4D5-encodingphagemid, pV0350-4, as template, and described as Lib-3 (see Lee et al.,2004. J Mol Biol 340:1073-1093). For initial selection with the naïvelibraries, hZ-, hA-, mZ, and mA-NSP4 were each immobilized separately onMaxisorp immunoplates (Nunc) and phage libraries were cycled through 4rounds of binding selection (see Lee et al., J Mol Biol, 2004,340:1073-1093) under high salt conditions in phosphate buffered salinesupplemented with 1% BSA or casein (BSA at rounds 1 and 3, casein atrounds 2 and 4), 0.1% Tween-20, and an additional 0.5 M NaCl to reducenon-specific interaction between the positively charged NSP4 proteinsand the negatively charged phage particles. Random clones selected fromrounds 3 and 4 were picked and assayed to identify specific bindersusing phage enzyme-linked immunosorbent assay (ELISA). The VH regions ofselected clones that bound to NSP4 were amplified by polymerase chainreaction (PCR) for sequencing.

Competition Phage ELISA

Phage clones were propagated from single colonies by growing in 30 ml2YT culture supplemented with carbenicillin and KO7 helper phageovernight at 30° C., purified, and assayed as described (see Lee et al.,J Mol Biol, 2004, 340:1073-1093). Phage at sub-saturating concentrationswere first incubated with increasing concentrations of target NSP4antigen (either hZ, hA, mZ, or mA) for 1 to 2 hour(s), and thentransferred to wells coated with the same NSP4 antigen to capture theunbound phage. The amount of phage bound was measured with anti-M13antibody-horseradish peroxidase (HRP) conjugate (GE Healthcare),developed using the substrate tetramethylbenzidine (TMB) (Kirkegaard andPerry Laboratories) for approximately 5 min, quenched with 1.0 M H3PO4,and read with a spectrophotometer at 450 nm wavelength as previouslydescribed (see Lee et al., J Mol Biol, 2004, 340:1073-1093). Inhibitoryconcentration (IC50) values were calculated as the concentration ofsoluble antigen that inhibited 50% of the phage binding to theimmobilized antigen.

Anti-NSP4 Antibody Production and Affinity Measurement

To generate IgG proteins for characterization, the variable domains ofselected phage clones were cloned into pRK5-based plasmid with humanlight chain or heavy chain (human IgG1) constant domain for transientIgG expression in Chinese hamster ovary (CHO) cells, and purified usingprotein A affinity chromatography.

Biolayer interferometry measurements (ForteBio) were used to determinethe affinity of anti-NSP4 antibodies against all four NSP4 antigens(hZ-, hA-, mZ-, and mA-NSP4). 5 μg/ml of each anti-NSP4 candidate wasimmobilized onto anti-human Fc sensors, and incubated with increasingconcentrations of each NSP4 antigens to obtain affinity measurements.These antibody candidates were then binned to one of the followingcategories: 1) Conformation and species-specific: binds to either hZ,hA, or mA (no mZ-specific binders were identified); 2)Conformation-specific: binds to active (hA/mA) or zymogen (hZ/mZ) formsof NSP4; 3) Species-specific: binds to human (hZ/hA) or mouse (mZ/mA)forms of NSP4; and 4) Pan-NSP4: binds to all four forms of NSP4(hZ/hA/mZ/mA).

Screening for NSP4 Blocking Antibodies

NSP4 fluorogenic activity assay was devised by screening a panel ofinternally-quenched fluorogenic peptide substrates (see Eigenbrot etal., Structure, 2012, 20:1040-1050) with NSP4. 50 nM of NSP4 wasincubated with 2.5 uM of each fluorescence-quenched peptide substrate in50 mM Tris pH 8.0, 200 mM NaCl, and 0.25% w/w CHAPS and the fluorescenceintensity determined using the SpectraMax M5 spectrophotometer(MolecularDevices) with excitation at 328 nm and emission at 393 nm. Thepeptide substrate cleaved most efficiently by NSP4 wasMca-Ile-Arg-Arg-Ser-Tyr-Ser-Phe-Lys[Dnp]-Lys, where Mca is7-methoxycoumarin-4-acetate and Dnp is dinitrophenol. Incubation withNSP4 resulted in a specific cleavage resulting in two distinctfragments: Mca-Ile-Arg-Arg and Ser-Tyr-Ser-Phe-Lys[Dnp]-Lys.

To identify blocking anti-NSP4 antibody candidates, 10 nM of recombinanthuman or mouse NSP4 was incubated with 500 nM of each antibody candidatefor 30 min at 37° C. This NSP4:antibody mixture was then mixed with 6.7uM of the fluorogenic peptide substrate, incubated for 5 min at 37° C.,and then read on the SpectraMax M5 spectrophotometer using theparameters described above.

Results

Three antibody candidates that strongly bound to NSP4 during heavy chainvariable domain (VH) library panning were isolated and used to generateIgG1 antibodies for further characterization (FIG. 13). Antibodycandidates 5-2, 5-3, and 5-4 demonstrated a high degree of bindingspecificity against active mouse (mA) NSP4 antigen (FIGS. 14A, 14B, and14C, respectively). Evaluation of the antibody candidates 5-2, 5-3, and5-4 demonstrated their ability to block peptide cleavage activity ofmouse NSP4 (FIG. 15). Furthermore, these antibodies did not blockpeptide cleavage activity of human NSP4, indicating that 5-2, 5-3, and5-4 were species specific to mouse NSP4 (FIG. 15).

Antibodies against human NSP4 can be generated using the methodsdescribed herein.

ANTIBODY SEQUENCES Heavy chain CDR1 for antibody 5-2′GFTFSNTYIS(SEQ ID NO: 1) Heavy chain CDR2 for antibody 5-2′GFIYPANGATYYADSVKG(SEQ ID NO: 2) Heavy chain CDR3 for antibody 5-2′RRYRLSFDY(SEQ ID NO: 3) Heavy chain CDR1 for antibody 5-3′GFTFSGNDIS(SEQ ID NO: 4) Heavy chain CDR2 for antibody 5-3′AGISPYGGSTYYADSVKG(SEQ ID NO: 5) Heavy chain CDR3 for antibody 5-3′RRVSFYSRHAGMDY(SEQ ID NO: 6) Heavy chain CDR1 for antibody 5-4′GFTFTSYAIS(SEQ ID NO: 7) Heavy chain CDR2 for antibody 5-4′AGISPSNGYTNYADSVKG(SEQ ID NO: 8) Heavy chain CDR3 for antibody 5-4′RAGRWTHSDIDY(SEQ ID NO: 9)Light chain CDR1 for antibodies 5-2′, 5-3′, and 5-4′RASQDVSTAVA (SEQ ID NO: 10)Light chain CDR2 for antibodies 5-2′, 5-3′, and 5-4′SASFLYS(SEQ ID NO: 11) Light chain CDR3 for antibodies 5-2′, 5-3′, and5-4′ QQSYTTPPT(SEQ ID NO: 12)Heavy chain variable region for antibody 5-2′EVQLVESGGGLVQPGGSLRLSCAASGFTFSNTYISWVRQAPGKGLEWVGFIYPANGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRRYRLSFDYWGQGTLVTVSS(SEQ ID NO: 13)Heavy chain variable region for antibody 5-3′EVQLVESGGGLVQPGGSLRLSCAASGFTFSGNDISWVRQAPGKGLEWVAGISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRRVSFYSRHAGMDYWGQGTLVTVSS(SEQ ID NO: 14)Heavy chain variable region for antibody 5-4′EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYAISWVRQAPGKGLEWVAGISPSNGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRAGRWTHSDIDYWGQGTLVTVSS(SEQ ID NO: 15)Light chain variable region for antibodies 5-2′, 5-3′, and 5-4′DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYTTPPTFGQGTKVEIKR (SEQ ID NO: 16)

Example 4: Characterization of NSP4 mRNA and Protein Expression Levels

Both mRNA and protein expression levels for NSP4 were measured invarious mouse cell types to profile the cell types for the presence ofNSP4.

Methods

RT-qPCR Measurement of NSP4 mRNA Levels.

To quantify the mRNA expression levels of NSP4 in immune cellpopulations from mouse femoral bone marrow, RT-qPCR analysis wasperformed. Immune cell populations from mouse femoral bone marrow wereidentified using standard flow cytometry protocols and the followingantibody clones: anti-CD11b (M1/70); anti-CD11c (N418); anti-B220(RA3-6B2); (BM8); anti-Ly6C (HK1.4); anti-Ly6G (1A8); anti-Siglec F(E50-2440). All antibodies were obtained from eBioscience. Viability wasevaluated using Sytox Blue (Invitrogen). Stained cells were sorted usinga BD Biosciences FACSAria cell sorter. The following populations weresorted for downstream analysis: B cells, B220+CD11b−; Monocytes,Ly6C+CD11b+; Neutrophils, Ly6G+CD11b+; Eosinophils, Siglec F+CD11b+.Total RNA content was isolated from each cell type using RNeasy Mini kit(Qiagen) and the corresponding cDNA were synthesized using the iScriptreverse transcriptase (Bio-Rad), all performed according to manufacturerinstructions. qPCR measurements were performed from cDNA samples usingthe TaqMan 2×PCR master mix (Applied Biosystems) with the followingthree NSP4/Prss57 primer/probe sets (Applied Biosystems): Prss57_1 (ABIMm01144794_m1), Prss57_2 (ABI Mm01144795_m1), and Prss57_3 (ABIMm01144796_m1). Gene expression levels are expressed as 2^(−dCt)relative to mouse 18S rRNA control.

Western Blot Detection of NSP4 Protein.

To measure for the presence of NSP4 protein in immune cell populationsfrom mouse femoral bone marrow, Western blot analysis was performedusing rabbit anti-mouse NSP4 polyclonal antibody. Bone-marrow derivedneutrophils were isolated from total bone marrow cells using the mouseneutrophil negative selection kit (Miltenyi Biotec) as performedaccording to manufacturer instructions. Bone marrow-derived eosinophilswere isolated and cultured from bone marrow cells as described in Dyeret al., 2008. J Immunol 181(6):4004-4009. Bone marrow-derived mast cellswere isolated and cultured from bone marrow cells as described in Lukacset al., 1996, Blood 87(6):2262-2268. Bone marrow-derived macrophageswere isolated from bone marrow cells as described in Zhang et al., 2008.Curr Protoc Immunol, Unit 14.1. Cell lysates were generated usingSDS-Laemmli sample buffer (Bio-Rad) and the concentrations weredetermined using BCA protein assay (Pierce).

Results

RT-qPCR Measurement of NSP4 mRNA Levels.

RT-qPCR analysis was performed to measure the mRNA expression levels ofNSP4 in immune cell populations isolated from mouse femoral bone marrow.RT-qPCR measurements using three different NSP4/Prss57 primer/probe setsthat span three different exon junctions corroborate NSP4 mRNAexpression levels in B cells, neutrophils, eosinophils, and monocytes(FIG. 16). As indicated by FIG. 16A-C, NSP4 mRNA was very highlyexpressed in eosinophils and measurements indicated the presence of NSP4mRNA expression in monocytes. NSP4 transcript was very low in fullydifferentiated neutrophils because transcription of NSP genes occurprimarily during the premyelocytic stage and cease in fullydifferentiated neutrophils. See Theilgaard-Monch et al., Blood105:1785-1796, 2005.

Western Blot Detection of NSP4 Protein.

Western blot analysis was performed using a rabbit anti-mouse NSP4polyclonal antibody to measure the protein expression levels of NSP4 inneutrophils, eosinophils, mast cells and macrophages isolated andcultured from mouse femoral bone marrow (FIG. 17). As indicated, NSP4protein (about 30 kD) was detected in neutrophils and eosinophilsisolated from the wild-type mice, but NSP4 was not detected in mastcells or macrophages from wild-type mice. NSP4 was not detected in cellsfrom NSP4-deficient mice.

Example 5: Neutrophil Recruitment Requires NSP4

In order to confirm the lack of neutrophil joint infiltration inNSP4^(−/−) mice, neutrophil recruitment was monitored by luminol-basedbioluminescence imaging in wild-type and NSP4^(−/−) mice.

Methods

Luminol-Bioluminescence Imaging of Neutrophil Myeloperoxidase Activityin Mouse Paws.

Myeloperoxidase (MPO) activity was monitored in vivo throughnon-invasive bioluminescence imaging (BLI) of the mouse paws. Mice wereanesthetized by 2% isoflurane (Butler Schein, 1 L/min flow), implantedwith a tail vein catheter, and positioned on a heated stage inside thePhoton Imager (Biospace Lab, Paris, France). Mice were injected throughthe tail vein catheter with a cocktail of 75 ul K/B×N serum and 150 ulMPO-sensitive luminol (Sigma-Aldrich). Light emission was recorded over8 minutes post injection of the cocktail at 5 minute, 6 hour, and 24hour intervals for each mouse. Bioluminescent pseudo-color images wereshown superimposed on a gray-scale bright field image of the mouse, withthe most intense signal being red and weakest signal blue. Forquantitative analysis of the luminol signal, an elliptical region ofinterest (ROI) was drawn on the bioluminescent images over all fourpaws. The area of the ROI was kept constant and results expressed asphoton counts per minute per cm² using the M3 Vision software (BiospaceLab).

To verify the expression of myeloperoxidase in NSP4-deficient mice,neutrophils from femoral bone marrow were isolated using the mouseneutrophil isolation kit (Miltenyi Biotec), and total cell lysates weremade using 2× Laemmli buffer (Bio-Rad). Myeloperoxidase and actin weredetected using anti-myeloperoxidase mouse monoclonal antibody (clone392105; R&D Biosystems) and anti-beta-actin mouse monoclonal antibody(clone 8H10D10; Cell Signaling Technology), respectively.

Results

Luminol-based bioluminescence imaging of neutrophil myeloperoxidase(MPO) activity was performed to image neutrophils in mouse paws (FIG.18A). NSP4-deficient neutrophils express normal levels of MPO (FIG.18B), therefore luminol-bioluminescence levels can be used as asurrogate marker for monitoring neutrophil recruitment. See Gross etal., 2009, Nat. Med. 15::455-461. NSP4^(−/−) mice exhibited asignificantly reduced neutrophil myeloperoxidase activity 24 h afterK/B×N serum transfer (FIG. 18C), suggesting a critical role for NSP4 inmediating neutrophil transmigration and joint infiltration in vivo.These optical imaging results are consistent with the lack of neutrophilinfiltration observed in the histological examinations described above,thus confirming the lack of neutrophil joint infiltration in NSP4^(−/−)mice.

Example 6: Affinity Improvement of Antibodies 3-5 and 5-1

Antibody clones 3-5 and 5-1 were affinity matured by optimizing theirantibody light chain sequence to bind with higher affinity to NSP4.

Methods

Library Construction for Affinity Improvement of Clones 3-5 and 5-1Derived from the VH Library.

Antibody clones Ab 3-5 (abbreviated Ab35) and Ab 5-1 (abbreviated Ab51)were selected for further affinity improvement. Phagemid pW0703 (derivedfrom phagemid pV0350-2b (Lee et al., J. Mol. Biol 340, 1073-1093(2004)), containing stop codon (TAA) in all CDR-L3 positions anddisplaying monovalent Fab on the surface of M13 bacteriophage served asthe library template for grafting heavy chain variable domains (VH) ofclones of interest from the VH library for affinity maturation. Bothhard and soft randomization strategies were used for affinitymaturation. For hard randomization, one light chain library withselected positions of the three light chain CDRs was randomized usingamino acids designed to mimic natural human antibodies and the designedDNA degeneracy was as described in Lee et al. (J. Mol. Biol 340,1073-1093 (2004)). For soft randomization, residues at positions 91-96of CDR-L3, 30-33, 35 of CDR-H1, 50, 52, 53-54, 56, and 58 of CDR-H2,95-100, 100A, and 100C of CDR-H3, were targeted; and three differentcombinations of CDR loops, H1/L3, H2/L3, and H3/L3, were selected forrandomization. To achieve the soft randomization conditions, whichintroduced the mutation rate of approximately 50% at the selectedpositions, the mutagenic DNA was synthesized with 70-10-10-10 mixturesof bases favoring the wild type nucleotides (Gallop et al., Journal ofMedicinal Chemistry 37:1233-1251 (1994)).

Phage Sorting Strategy to Generate Affinity Improvement.

For affinity improvement selection, phage libraries were subjected toone round of plate sorting followed by three additional rounds ofsolution sorting with increasing stringency. For round one, platesorting strategy was used, where 3 OD/ml in 1% BSA and 0.05% Tween 20 ofphage input were incubated with 5 ug/ml of mouse NSP4 at 100 ul/wellbuffer containing 1% BSA and 0.05% Tween-20 for 1.5 h at roomtemperature with gentle shaking. The wells were washed with PBS-0.05%Tween-20 ten times. Bound phage was eluted with 150 μl/well 50 mM HCl,500 mM KCl for 30 minutes, and subsequently neutralized by 50 μl/well of1M Tris pH 8, titered, and propagated for the next round. For rounds twothrough four, solution sorting strategies were used with increasingselection stringency. Specifically, biotinylated mouse NSP4 were used at20 nM (round 2), 5 nM (round 3), and 0.5 nM with 500 nM unlabeled mNSP4competitor (round 4) to select for improved on-rate and off-ratebinders. To determine background binding, control wells containing phagewere captured on neutravidin-coated plates. Bound phage captured onneutravidin-coated wells was eluted with 150 μl/well 50 mM HCl, 500 mMKCl for 30 minutes, and subsequently neutralized by 50 μl/well of 1MTris pH8, titered, and propagated for the next round.

Epitope Mapping.

Recombinant mouse NSP4 was preincubated with buffer alone, NSP4 antibody5-1 (Fab format), or with 10 ug/ml heparin sulfate (Sigma Aldrich) for15 minutes at room temperature in phosphate buffered saline. 50 nM ofthis mouse NSP4 mixture was then incubated with optical sensors coatedwith NSP4 antibodies that were immobilized at 40 ug/ml in 1× KineticBuffer (Pall Corporation). The binding was measured using Octet biolayerinterferometry (Pall Corporation).

Results

Multiple antibody clones were tested in a phage IC50 experiment toestimate their binding affinity to NSP4. Briefly, 2 ug/ml of NSP4 (hZ,hA, mZ, or mA variants) was coated on wells overnight at 4° C. in PBSsupplemented with 0.5% BSA. Each individual phage clone (at 0.01 OD/ml)was purified and incubated with increasing concentrations of solubleNSP4 protein for 2 h at room temperature before being placed into wellscoated with immobilized NSP4 protein for 15 min at room temperature. Thewells were then washed with PBS supplemented with 0.05% Tween-20 for 10times and developed using anti-M13 phage antibody conjugated with HRP(New England Biolabs). As shown in FIG. 19, higher affinity phage cloneswould more likely remain associated with the soluble antigen at lowerantigen concentrations than lower affinity phage clones, resulting inless binding to the immobilized antigen coated on the wells during the15 min incubation period. The soluble phage:antigen complex, with higherlikelihood to be high-affinity phage clones, would thus be removedthrough successive washing steps, resulting in lower A450 signal at anygiven concentration of soluble antigen. Consequently, higher affinityphage clones would have a lower phage IC50 value, with a concomitantleftward-shift in the binding curves, compared to lower affinity phageclones. The HVR sequences for these clones are provided in Tables 3 and4 below. Each of the antibodies in Tables 3 and 4 used the same lightchain, which included a light chain variable region corresponding to SEQID NO: 16 and HVR-L1, HVR-L2, and HVR-L3 sequences corresponding to SEQID NO:19, 11, and 12, respectively.

TABLE 3 HC HVR Sequences for Anti-NSP4 Antibodies. SEQ SEQ SEQ CloneHVR-H1 ID HVR-H2 ID HVR-G3 ID 1-1 GFTFSGSWIS 20 GTISPYNGSTYYADSVKG 21RVLRPKVYASVMDY 22 1-2 GFTFSGYSIH 23 AGISPTNGYTDYADSVKG 24 RLVFYRGVMDY 251-3 GFTFSDNWIS 26 GYIYPASGYTDYADSVKG 27 SDSPHAYWYAMDY 28 1-5 GFTFTNNSIS29 GAISPNNGSTYYADSVKG 30 RNAWHYSWVGVMDY 31 2-1 GFTFTDYSIH 32AEIYPYSGDTYYADSVKG 33 RDGDGWFDWAMDY 34 2-2 GFTFSSTAIS 35GEIYPSDGYTDYADSVKG 36 RVKWAVSSLGVMDY 37 2-3 GFTFTDSDIS 38AWISPSDGATDYADSVKG 39 HEASDDDYAIDY 40 2-4 GFTFSDYWIS 41AGISPNNGDTYYADSVKG 42 REDDDERDYAMDY 43 2-5 GFTFTGYGIS 44GWIYPASGATYYADSVKG 45 RHRAFDWYPYYIGSSVMDY 46 3-2 GFTFSDYSIS 47GEINPAGGATYYADSVKG 48 RGDFPFWSDAYYVMDY 49 3-5 GFTFSDNDIS 50GSISPDNGDTNYADSVKG 51 RDDVPAVFTSAMDY 52 4-2 GFTFSGSDIS 53GEIYPSNGDTYYADSVKG 54 RSVRPSWWAMDY 55 4-3 GFTFSSYDIS 56GTISPYDGYTDYADSVKG 57 RYIRRYSVHYGMDY 58 4-4 GFTFTSTSIH 59AEITPHGGYTNYADSVKG 60 RGRTKWGWLYGMDY 61 4-5 GFTFTNNSIH 62AEIAPDDGYTYYADSVKG 63 RGVIRYAYLYAMDY 64 5-1 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 KSLFHNVAFDY 67 5-2 GFTFSNTYIS 1 GFIYPANGATYYADSVKG2 RRYRLSFDY 3 5-3 GFTFSGNDIS 4 AGISPYGGSTYYADSVKG 5 RRVSFYSRHAGMDY 6 5-4GFTFTSYAIS 7 AGISPSNGYTNYADSVKG 8 RAGRWTHSDIDY 9

TABLE 4Heavy Chain Variable Region Sequences for Anti-NSP4 Antibodies. CDRsequences are underlined. SEQ Clone HC Variable Region Sequences ID 1-1EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSWISWVRQAPGKG 68LEWVGTISPYNGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVLRPKVYASVMDYWGQGTLVTVSS 1-2EVQLVESGGGLVQPGGSLRLSCAASGFTFSGYSIHWVRQAPGKG 69LEWVAGISPTNGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARLVFYRGVMDYWGQGTLVTVSS 1-3EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNWISWVRQAPGK 70GLEWVGYIYPASGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCASDSPHAYWYAMDYWGQGTLVTVSS 1-5EVQLVESGGGLVQPGGSLRLSCAASGFTFTNNSISWVRQAPGKG 71LEWVGAISPNNGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARNAWHYSWVGVMDYWGQGTLVTVSS 2-1EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYSIHWVRQAPGKG 72LEWVAEIYPYSGDTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDGDGWFDWAMDYWGQGTLVTVSS 2-2EVQLVESGGGLVQPGGSLRLSCAASGFTFSSTAISWVRQAPGKG 73LEWVGEIYPSDGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARVKWAVSSLGVMDYWGQGTLVTVSS 2-3EVQLVESGGGLVQPGGSLRLSCAASGFTFTDSDISWVRQAPGKG 74LEWVAWISPSDGATDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAHEASDDDYAIDYWGQGTLVTVSS 2-4EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWISWVRQAPGK 75GLEWVAGISPNNGDTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAREDDDERDYAMDYWGQGTLVTVSS 2-5EVQLVESGGGLVQPGGSLRLSCAASGFTFTGYGISWVRQAPGKG 76LEWVGWIYPASGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARHRAFDWYPYYIGSSVMDYWGQGTLVTVSS 3-2EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYSISWVRQAPGKG 77LEWVGEINPAGGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGDFPFWSDAYYVMDYWGQGTLVTVSS 3-5EVQLVESGGGLVQPGGSLRLSCAASGFTFSDNDISWVRQAPGKG 78LEWVGSISPDNGDTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARDDVPAVFTSAMDYWGQGTLVTVSS 4-2EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSDISWVRQAPGKG 79LEWVGEIYPSNGDTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARSVRPSWWAMDYWGQGTLVTVSS 4-3EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYDISWVRQAPGKG 80LEWVGTISPYDGYTDYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARYIRRYSVHYGMDYWGQGTLVTVSS 4-4EVQLVESGGGLVQPGGSLRLSCAASGFTFTSTSIHWVRQAPGKG 81LEWVAEITPHGGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGRTKWGWLYGMDYWGQGTLVTVSS 4-5EVQLVESGGGLVQPGGSLRLSCAASGFTFTNNSIHWVRQAPGKG 82LEWVAEIAPDDGYTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARGVIRYAYLYAMDYWGQGTLVTVSS 5-1EVQLVESGGGLVQPGGSLRLSCAASGFTFSGSGIHWVRQAPGKG 83LEWVAWISPTGGNTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCAKSLFHNVAFDYWGQGTLVTVSS 5-2EVQLVESGGGLVQPGGSLRLSCAASGFTFSNTYISWVRQAPGKG 84LEWVGFIYPANGATYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRYRLSFDYWGQGTLVTVSS 5-3EVQLVESGGGLVQPGGSLRLSCAASGFTFSGNDISWVRQAPGKG 85LEWVAGISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRVSFYSRHAGMDYWGQGTLVTVSS 5-4EVQLVESGGGLVQPGGSLRLSCAASGFTFTSYAISWVRQAPGKG 86LEWVAGISPSNGYTNYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARAGRWTHSDIDYWGQGTLVTVSS

FIG. 19 demonstrates the results of the phage IC50 experiments, whichused soluble hZ-, hA-, mZ-, or mA-NSP4 as a competitor (as labeled). Inthese experiments, higher affinity clones were bound to soluble NSP4 atlower soluble NSP4 concentrations and thus have curves shifted to theleft. Based on these results, individual antibodies were purified andexpressed for further characterization. These results are summarized inthe table below.

TABLE 8 Summary of phage IC50 values of NSP4-specific antibody phageclones. Soluble competitor used for IC50 (nM) Ab clones hZ (NSP4) hA(NSP4) mZ (NSP4) mA (NSP4) 1-series (panned against hZ-NSP4) 1-1 154.70— — — 1-2 22.08 — — — 1-3 52.05 — — — 1-5 114.50 — — — 2-series (pannedagainst hZ-NSP4) 2-1 7.62 3.81 — — 2-2 10.42 10.11 — — 2-3 3.07 4.96 — —2-4 2.30 1.63 — — 2-5 21.98 5130.00 — — 3-series (panned againstmZ-NSP4) 3-2 — — 0.15 — 3-5 — — 0.18 — 4-series (panned against hA-NSP4)4-2 — 84.76 — — 4-3 — 1.58 — — 4-4 — 1.48 — — 5-series (panned againstmA-NSP4) 5-1 — — — 9.93 5-2 — — — 25.01 5-3 — — — 319.00 5-4 — — — 60.48

These antibodies were further characterized using biolayerinterferometry measurements to estimate affinity (kD) to different NSP4proteins (i.e., hZ, hA, mZ, and mA, as labeled). FIGS. 20A and B showthe results for antibodies 1-1 and 1-2, respectively. FIGS. 21A and Bshow the results for antibodies 1-3 and 1-5, respectively. FIGS. 22A-Dshow the results for antibodies 2-1, 2-2, 2-3 and 2-4, respectively.FIG. 23 shows the results for antibody 2-5. FIGS. 24A and B show theresults for antibodies 3-2 and 3-5, respectively. FIGS. 25A-C show theresults for antibodies 4-2, 4-3 and 4-4, respectively. FIGS. 26A-D showthe results for antibodies 5-1, 5-2, 5-3 and 5-4, respectively. Thesebinding data suggest a panel of antibodies with distinct NSP4-bindingcharacteristics, ranging from antibodies that are highlyconformational-specific and species-specific (e.g., specific for one ofhZ-NSP4, hA-NSP4, or mA-NSP4 such as antibodies 1-2, 4-3, and 5-3), toantibodies that are conformational-specific but not species-specific(e.g., specific for zymogen-NSP4 or active-NSP4 such as antibodies 2-2,2-5, and 5-1), to antibodies that are species-specific but notconformational-specific (e.g., specific for human-NSP4 or mouse-NSP4such as antibodies 3-2, 4-2, and 4-4), and finally to antibodies thatare pan-NSP4 binders (e.g., bind to human and mouse NSP4 in theirzymogen and active conformational states such as antibodies 2-1, 2-3,2-4, and 3-5).

Antibodies were further tested for the ability to block NSP4 enzymaticactivity. The ability to block human and mouse NSP4 was tested. FIG. 27shows the effect of antibodies on zymogen NSP4. FIG. 28 shows the effectof antibodies on active NSP4. As shown in FIG. 28, antibodies 4-3 and4-4 blocked human NSP4 activity, whereas each of the 5-series antibodiesblocked mouse NSP4 activity. The in vitro properties of the antibodiesare summarized in FIG. 29.

Without wishing to be bound to theory, it was hypothesized that the5-series antibodies, due to their ability to block NSP4 enzyme activityand their preferential binding to the active form but not the zymogenform of NSP4, bound NSP4 proximal to the enzyme active site, a regionthat undergoes significant conformational change during the proteaseactivation process. In contrast, it was hypothesized that the 3-seriesantibodies, due to their inability to block NSP4 enzyme activity andtheir conformational-insensitivity, bound NSP4 distal to the enzymeactive site. This hypothesis on antibody epitopes was confirmed in aseries of experiences depicted in FIGS. 30 and 31.

As shown in FIG. 30, Fab 5-1 interferes with Ab5-1, 5-2, and 5-4binding, but not with Ab 3-2 or 3-5 binding, to mouse NSP4. Similarly,as shown in FIG. 31, heparin has little effect on Ab 5-1, 5-2, and 5-4binding, but was able to significantly diminish binding of Ab 3-2 and3-5 to NSP4. These results suggest that Ab 5-1, 5-2, 5-4 bind near theenzyme active site; in contrast, Ab 3-2 and 3-5 bind near the heparinbinding site away from the enzyme active site.

Antibody clones 3-5 (referred to as Ab35 below) and 5-1 (referred to asAb51 below) were chosen for further affinity improvement (carried out asdescribed above). Ab35 was chosen due to its ability to bind to bothhuman and mouse NSP4 and both zymogen and active conformations. Ab51 waschosen for its ability to inhibit mouse NSP4 catalytic activity withhighest potency among the 5-series antibodies.

Tables 5 and 6 list affinity matured variants of antibody 3-5 (called35.WT in the tables below), labeled as 35.XX, and affinity maturedvariants of antibody 5-1 (called 51.WT in the tables below), labeled as51.XX. Heavy chain HVR sequences are listed in Table 5; light chain HVRsequences are listed in Table 6.

TABLE 5 HC HVR Sequences for Affinity Matured Anti-NSP4 Antibodies. SEQSEQ SEQ Clone HVR-H1 ID HVR-H2 ID HVR-H3 ID 35.WT GFTFSDNDIS 50GSISPDNGDTNYADSVKG 51 RDDVPAVFTSAMDY 52 35.14 GFTFSDNDIS 50GSISPDNGDTNYADSVKG 51 RDDVPAVFTSAMDY 52 35.50 GFTFSDNDIS 50GSISPDNGDTNYADSVKG 51 RDDVPAVFTSAMDY 52 35.62 GFTFSDNDIS 50GSISPDNGDTNYADSVKG 51 RDDVPAVFTSAMDY 52 35.77 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 KRHLHNVAFDY 87 51.WT GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 KSLFHNVAFDY 67 51.30 GFTFSGSGIH 65AWIPTAGGNTYYADSVKG 88 KSLFHNVAFDY 67 51.50 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 KSLFHNVAFDY 67 51.51 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 RGLFHNVAFDY 89 51.59 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 RVFFHNVAFDY 90 51.72 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 KSLFHNVAFDY 67 51.82 GFTFSGSGIH 65AWISPTGGNTYYADSVKG 66 RGLFHNVAFDY 89 Consensus X₁X₂X₃FHNVAFDY 91

TABLE 6 LC HVR Sequences for Affinity Matured Anti-NSP4 Antibodies.Clone HVR-L1 SEQ ID HVR-L2 SEQ ID HVR-L3 SEQ ID 35.WT RASQDVS 19 SASFLYS11 QQSYTTPPT 12 35.14 RASQDVS 19 SASFLYS 11 QQSYGFPLT 92 35.50 RASQDVS19 SASFLYS 11 QQSYDFPLT 93 35.62 RASQDVS 19 SASFLYS 11 QQSAGFPLT 94Consensus QQSX₁X₂X₃PX₄T 95 35.77 RASQDVS 19 SASFLYS 11 QQAYSAPPT 9651.WT RASQDVS 19 SASFLYS 11 QQSYTTPPT 12 51.30 RASQDVS 19 SASFLYS 11QQSYTAPPT 97 51.50 RASQDVS 19 SASFLYS 11 QQANSTPPT 98 51.51 RASQDVS 19SASFLYS 11 QQSYTAPPT 97 51.59 RASQDVS 19 SASFLYS 11 QQNFSSPPT 99 51.72RASQDVS 19 SASFLYS 11 QQSYTAPPT 97 51.82 RASQDVS 19 SASFLYS 11 QQTYNAPPT100 Consensus QQX₁X₂X₃X₄PPT 101

Table 7 lists heavy chain and light chain variable region sequences foraffinity matured variants of antibody 3-5 (called 35.WT in the tablebelow), labeled as 35.XX, and affinity matured variants of antibody 5-1(called 51.WT in the table below), labeled as 51.XX. Constant regionsequences are provided below the table.

TABLE 7HC and LC Variable Region Sequences for Affinity Matured Anti-NSP4Antibodies. CDR sequences are underlined. SEQ SEQ CloneHC Variable Region Sequences ID LC Variable Region Sequences ID 35.WTEVQLVESGGGLVQPGGSLRLSCAAS 78 DIQMTQSPSSLSASVGDRVTIT 16GFTFSDNDISWVRQAPGKGLEWVG CRASQDVSTAVAWYQQKPGK SISPDNGDTNYADSVKGRFTISADTSAPKLLIYSASFLYSGVPSRFSGS KNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYDDVPAVFTSAMDYWGQGTLVTVSS CQQSYTTPPTFGQGTKVEIKR 35.14EVQLVESGGGLVQPGGSLRLSCAAS 78 DIQMTQSPSSLSASVGDRVTIT 102GFTFSDNDISWVRQAPGKGLEWVG CRASQDVSTAVAWYQQKPGK SISPDNGDTNYADSVKGRFTISADTSAPKLLIYSASFLYSGVPSRFSGS KNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYDDVPAVFTSAMDYWGQGTLVTVSS CQQSYGFPLTFGQGTKVEIKR 35.50EVQLVESGGGLVQPGGSLRLSCAAS 78 DIQMTQSPSSLSASVGDRVTIT 103GFTFSDNDISWVRQAPGKGLEWVG CRASQDVSTAVAWYQQKPGK SISPDNGDTNYADSVKGRFTISADTSAPKLLIYSASFLYSGVPSRFSGS KNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYDDVPAVFTSAMDYWGQGTLVTVSS CQQSYDFPLTFGQGTKVEIKR 35.62EVQLVESGGGLVQPGGSLRLSCAAS 78 DIQMTQSPSSLSASVGDRVTIT 104GFTFSDNDISWVRQAPGKGLEWVG CRASQDVSTAVAWYQQKPGK SISPDNGDTNYADSVKGRFTISADTSAPKLLIYSASFLYSGVPSRFSGS KNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYDDVPAVFTSAMDYWGQGTLVTVSS CQQSAGFPLTFGQGTKVEIKR 35.77EVQLVESGGGLVQPGGSLRLSCAAS 105 DIQMTQSPSSLSASVGDRVTIT 106GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCA GSGTDFTLTISSLQPEDFATYYKRHLHNVAFDYWGQGTLVTVSS CQQAYSAPPTFGQGTKVEIKR 51.WTEVQLVESGGGLVQPGGSLRLSCAAS 83 DIQMTQSPSSLSASVGDRVTIT 16GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCA GSGTDFTLTISSLQPEDFATYYKSLFHNVAFDYWGQGTLVTVSS CQQSYTTPPTFGQGTKVEIKR 51.30EVQLVESGGGLVQPGGSLRLSCAAS 107 DIQMTQSPSSLSASVGDRVTIT 108GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WIPTAGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCA GSGTDFTLTISSLQPEDFATYYKSLFHNVAFDYWGQGTLVTVSS CQQSYTAPPTFGQGTKVEIKR 51.50EVQLVESGGGLVQPGGSLRLSCAAS 83 DIQMTQSPSSLSASVGDRVTIT 109GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCA GSGTDFTLTISSLQPEDFATYYKSLFHNVAFDYWGQGTLVTVSS CQQANSTPPTFGQGTKVEIKR 51.51EVQLVESGGGLVQPGGSLRLSCAAS 110 DIQMTQSPSSLSASVGDRVTIT 108GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYGLFHNVAFDYWGQGTLVTVSS CQQSYTAPPTFGQGTKVEIKR 51.59EVQLVESGGGLVQPGGSLRLSCAAS 111 DIQMTQSPSSLSASVGDRVTIT 112GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYVFFHNVAFDYWGQGTLVTVSS CQQNFSSPPTFGQGTKVEIKR 51.72EVQLVESGGGLVQPGGSLRLSCAAS 83 DIQMTQSPSSLSASVGDRVTIT 108GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCA GSGTDFTLTISSLQPEDFATYYKSLFHNVAFDYWGQGTLVTVSS CQQSYTAPPTFGQGTKVEIKR 51.82EVQLVESGGGLVQPGGSLRLSCAAS 110 DIQMTQSPSSLSASVGDRVTIT 113GFTFSGSGIHWVRQAPGKGLEWVA CRASQDVSTAVAWYQQKPGK WISPTGGNTYYADSVKGRFTISADTAPKLLIYSASFLYSGVPSRFSGS SKNTAYLQMNSLRAEDTAVYYCAR GSGTDFTLTISSLQPEDFATYYGLFHNVAFDYWGQGTLVTVSS CQQTYNAPPTFGQGTKVEIKR

HC Constant Region Sequence (SEQ ID NO: 114)ASTKGPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCNVAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIFPPKIKDVLMISLSPIVTCVVVAVSEDDPDVQISWFVNNVEVHTAQTQTHREDYASTLRVVSALPIQHQDWMSGKEFKCKVNNKDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPGK LC Constant Region Sequence(SEQ ID NO: 115) ADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKS FNRNEC

The improvement of affinity-matured antibody clones was estimated in aseries of phage IC50 experiments. FIG. 32 shows the affinities ofaffinity-matured antibody clones from Ab35 towards human and mouse NSP4.FIG. 33 shows the affinities of affinity-matured antibody clones fromthe Ab51 towards human NSP4. As shown, the affinities of the Ab35 cloneswere not improved as much as the Ab51 clones. Without wishing to bebound to theory, this could be due to the fact that the Ab35 startedwith higher affinity. In contrast, the affinities of the Ab51 improvedsignificantly (over 500×).

Table 9 summarizes improvement of affinity-matured antibody clones asdetermined by their IC50 values in a phage competition binding assay.

TABLE 9 Affinity-matured antibodies. mNSP4 IC50 hNSP4 IC50 Affinityimprovement Ab Clones (nM) (nM) to mNSP4 35.WT 0.131 32.79 — 35.14 0.04823.41 2.7 35.50 0.075 22.75 1.7 35.62 0.206 25.69 0.6 35.77 0.064 — 2.051.WT 18.670 — — 51.30 0.116 — 160.9 51.50 0.105 — 178.0 51.51 0.296 —63.0 51.59 0.095 — 196.0 51.72 0.375 — 49.8 51.82 0.037 — 504.6

Ab35 and Ab51 were affinity improved up to 2.7-fold and 504.6-fold,respectively, over their parental clones by exploring and optimizing theCDR sequences on the antibody light chain for binding to NSP4. Bothaffinity matured Ab35 and Ab51 clones reached the targetedpicomolar-affinity to NSP4 and bound to NSP4 with distinct epitopes.Without wishing to be bound to theory, it is thought that Ab35-derivedclones bind to NSP4 at the heparin-binding site and Ab51-derived clonesbind to NSP4 proximal to its enzyme active site.

Example 7: NSP4 Catalytic Activity In Vivo

To confirm that the enzymatic activity of NSP4 is associated with thedisease phenotype observed in the K/B×N serum-transfer mouse model, theK/B×N model is compared in mice having wild-type NSP4, an NSP4 knockoutand a catalytically dead NSP4 knockin (e.g., mice having an S224Amutation in their NSP4 protein sequence). In each of these three mousestrains, serum-induced vascular permeability is monitored in vivo usingNIRF, luminol-bioluminescence imaging and histological examination. Ifthe mouse expressing the catalytically dead NSP4 protein showsprotection in the K/B×N model similar to the NSP4 knockout mouse, thisresult may indicate that the catalytic activity of NSP4 is associatedwith the disease phenotype and that inhibitors of NSP4 catalyticactivity (e.g., an anti-NSP4 antibody such as antibody 51.WT, 51.30,51.50, 51.51, 51.59, 51.72, 51.82) would be useful as therapeutic agentsfor various vascular and inflammatory disorders associated with NSP4activity.

Example 8: Inhibition of NSP4 Activity NSP4 in a Mouse Model

Anti-NSP4 antibodies are tested in a mouse K/B×N model to confirm thatantibody inhibition of NSP4 activity is protective of the diseasephenotype observed in the wildtype NSP4 K/B×N model. K/B×N mice arechallenged as described above either in the presence or absence of ananti-NSP4 antibody. NSP4 antibodies that either bind to the heparinbinding site (e.g., the 35 series antibodies 35.WT, 35.14, 35.50, 35.62and/or 35.77) or inhibit the catalytic activity of NSP4 (the 51 seriesantibodies 51.WT, 51.30, 51.50, 51.51, 51.59, 51.72, and/or 51.82) aretested in the K/B×N model. The results are compared to a controlantibody (e.g., an antibody that does not bind to NSP4). If the micetreated with the anti-NSP4 antibodies show protection in the K/B×N modelsimilar to the NSP4 knockout mouse, this result may indicate that theinhibition of NSP4 using an anti-NSP4 antibody would be useful as atherapeutic agent for various vascular and inflammatory disordersassociated with NSP4 activity.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, the descriptions and examples should not be construed aslimiting the scope of the invention. The disclosures of all patent andscientific literature cited herein are expressly incorporated in theirentirety by reference.

What is claimed is:
 1. An isolated nucleic acid encoding an anti-NSP4antibody comprising a heavy chain and a light chain, wherein the lightchain comprises an HVR-L1 comprising the sequence of SEQ ID NO:19, anHVR-L2 comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprisingthe sequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:56, an HVR-H2 comprising thesequence of SEQ ID NO:57, and an HVR-H3 comprising the sequence of SEQID NO:58.
 2. A host cell comprising the nucleic acid of claim
 1. 3. Amethod for producing an antibody comprising culturing the host cell ofclaim 2 under conditions suitable for production of the antibody.
 4. Themethod of claim 3, further comprising recovering the antibody producedby the host cell.
 5. A vector comprising the nucleic acid of claim
 1. 6.The isolated nucleic acid of claim 1, wherein the light chain comprisesa light chain variable region comprising the sequence of SEQ ID NO:16.7. The isolated nucleic acid of claim 1, wherein the heavy chaincomprises a heavy chain variable region comprising the sequence of SEQID NO:
 80. 8. The isolated nucleic acid of claim 1, wherein the antibodyis an antibody fragment selected from the group consisting of a Fab,Fab′-SH, Fv, scFv, and (Fab′)₂ fragment.
 9. The isolated nucleic acid ofclaim 1, wherein the antibody comprises a constant region of human IgG1,IgG2, IgG3, or IgG4.
 10. An isolated nucleic acid encoding an anti-NSP4antibody comprising a heavy chain and a light chain, wherein the lightchain comprises an HVR-L1 comprising the sequence of SEQ ID NO:19, anHVR-L2 comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprisingthe sequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:32, an HVR-H2 comprising thesequence of SEQ ID NO:33, and an HVR-H3 comprising the sequence of SEQID NO:34.
 11. A vector comprising the nucleic acid of claim
 10. 12. Ahost cell comprising the nucleic acid of claim
 10. 13. A method forproducing an antibody comprising culturing the host cell of claim 12under conditions suitable for production of the antibody.
 14. The methodof claim 13, further comprising recovering the antibody produced by thehost cell.
 15. An isolated nucleic acid encoding an anti-NSP4 antibodycomprising a heavy chain and a light chain, wherein the light chaincomprises an HVR-L1 comprising the sequence of SEQ ID NO:19, an HVR-L2comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprising thesequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:38, an HVR-H2 comprising thesequence of SEQ ID NO:39, and an HVR-H3 comprising the sequence of SEQID NO:40.
 16. A vector comprising the nucleic acid of claim
 15. 17. Ahost cell comprising the nucleic acid of claim
 15. 18. A method forproducing an antibody comprising culturing the host cell of claim 17under conditions suitable for production of the antibody.
 19. The methodof claim 18, further comprising recovering the antibody produced by thehost cell.
 20. An isolated nucleic acid encoding an anti-NSP4 antibodycomprising a heavy chain and a light chain, wherein the light chaincomprises an HVR-L1 comprising the sequence of SEQ ID NO:19, an HVR-L2comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprising thesequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:41, an HVR-H2 comprising thesequence of SEQ ID NO:42, and an HVR-H3 comprising the sequence of SEQID NO:43.
 21. A vector comprising the nucleic acid of claim
 20. 22. Ahost cell comprising the nucleic acid of claim
 20. 23. A method forproducing an antibody comprising culturing the host cell of claim 22under conditions suitable for production of the antibody.
 24. The methodof claim 23, further comprising recovering the antibody produced by thehost cell.
 25. An isolated nucleic acid encoding an anti-NSP4 antibodycomprising a heavy chain and a light chain, wherein the light chaincomprises an HVR-L1 comprising the sequence of SEQ ID NO:19, an HVR-L2comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprising thesequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:53, an HVR-H2 comprising thesequence of SEQ ID NO:54, and an HVR-H3 comprising the sequence of SEQID NO:55.
 26. A vector comprising the nucleic acid of claim
 25. 27. Ahost cell comprising the nucleic acid of claim
 25. 28. A method forproducing an antibody comprising culturing the host cell of claim 27under conditions suitable for production of the antibody.
 29. The methodof claim 28, further comprising recovering the antibody produced by thehost cell.
 30. An isolated nucleic acid encoding an anti-NSP4 antibodycomprising a heavy chain and a light chain, wherein the light chaincomprises an HVR-L1 comprising the sequence of SEQ ID NO:19, an HVR-L2comprising the sequence of SEQ ID NO:11, and an HVR-L3 comprising thesequence of SEQ ID NO:12; and wherein the heavy chain comprises anHVR-H1 comprising the sequence of SEQ ID NO:59, an HVR-H2 comprising thesequence of SEQ ID NO:60, and an HVR-H3 comprising the sequence of SEQID NO:61.
 31. A vector comprising the nucleic acid of claim
 30. 32. Ahost cell comprising the nucleic acid of claim
 30. 33. A method forproducing an antibody comprising culturing the host cell of claim 32under conditions suitable for production of the antibody.
 34. The methodof claim 33, further comprising recovering the antibody produced by thehost cell.